Method for producing an l-amino acid using a bacterium of the enterobacteriaceae family

ABSTRACT

A method for producing an L-amino acid is described using a bacterium of the Enterobacteriaceae family, wherein the bacterium contains a protein which is able to confer resistance to growth inhibition by L-cysteine.

This application is claims priority under 35 U.S.C. §119 to RussianPatent Application No. 2010101136, filed on Jan. 15, 2010, which isincorporated in its entirety by reference. The Sequence Listing inelectronic format filed herewith is also hereby incorporated byreference in its entirety (File Name: 2011-01-11T_US-454_Seq_List; FileSize: 31 KB; Date Created: Jan. 11, 2011).

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to the microbiological industry, andspecifically to a method for producing an L-amino acid using a bacteriumof the Enterobacteriaceae family which has a protein derived from abacterium belonging to the genus Pantoea, and the protein is able toconfer resistance to cysteine.

2. Description of the Related Art

Conventionally, L-amino acids are industrially produced by fermentationmethods utilizing strains of microorganisms obtained from naturalsources, or mutants thereof. Typically, the microorganisms are modifiedto enhance production yields of L-amino acids.

Many techniques to enhance L-amino acid production yields have beenreported, including transformation of microorganisms with recombinantDNA (U.S. Pat. No. 4,278,765). Other techniques for enhancing productionyields include increasing the activities of enzymes involved in aminoacid biosynthesis and/or desensitizing the target enzymes to feedbackinhibition by the resulting L-amino acid (U.S. Pat. Nos. 4,346,170;5,661,012; and 6,040,160).

A new microbial strain is disclosed which is suitable for thefermentative production of L-cysteine, L-cystine, N-acetyl-serine whichis produced from the non-enzymatic conversion of O-acetyl-L-serine,and/or thiazolidine derivatives. This new strain overexpresses at leastone gene which codes for a protein that mediates cellular clearance ofantibiotics or other substances that are toxic for the microorganism (EP0885962).

A chromosomal fragment has been identified in a gene bank fromEscherichia coli, which is able to increase the yield of cysteine in anindustrial production strain. Subcloning and genetic analysis showedthat an open reading frame coding for a product of 299 amino acids,called Orf299, was responsible. The Orf299 was synthesized in the T7polymerase/promoter system and exhibited the properties of an integralmembrane protein. These results further indicated that ORF299 codes foran export pump responsible for excreting different metabolites of thecysteine pathway (Dassler T. et al, Mol. Microbiol.; 36(5):1101-12(2000).

The ORF yfiK gene was discovered to be able to increase cysteineproduction when overexpressed in an industrial E. coli productionstrain. The yfiK gene product is an integral membrane protein with aboutsix predicted transmembrane helices, and it belongs to the RhtB familyof export proteins. YfiK overproduction from a plasmid leads to drasticand parallel secretion of O-acetyl-L-serine and cysteine into themedium, but only when the organism possesses a serine transacetylasethat is insensitive to feedback inhibition by cysteine. When excessO-acetyl-L-serine is added to the medium, this requirement for thepresence of a feedback-insensitive serine transacetylase during cysteinesecretion can be obviated both in the yfiK-carrying transformant and inthe wild-type strain. A delta yfiK mutant did not show any phenotype,and was able to export O-acetyl-L-serine and cysteine when transformedwith a plasmid carrying ydeD, a previously characterized, alternateO-acetyl-L-serine/cysteine exporter. Since an ydeD-yfiK double mutantshowed the same pattern, it appears that YfiK and YdeD actindependently. The necessity for the cell to regulate the size of theinternal pool of O-acetyl-L-serine via synthesis of exporter proteinscould be connected to the fact that this compound (when suppliedexternally) inhibits growth. Overexpression of either ydeD or yfiKalleviates this inhibition, and increases resistance to azaserine, whichis an analog of O-acetyl-L-serine (Franke I et. al., J. Bacteriol.;185(4):1161-6 (2003)).

Assembly of E. coli cytochrome bd and periplasmic cytochromes requiresthe ATP-binding cassette transporter CydDC, the substrate of which isunknown. Two-dimensional SDS-PAGE comparison of periplasm from wild-typeand cydD mutant strains revealed that the latter was deficient inseveral periplasmic transport binding proteins, even though no singlemajor protein was missing in the cydD periplasm. Instead, CydDC exportsfrom the cytoplasm to the periplasm the amino acid cysteine, which canbe further demonstrated by using reverted membrane vesicles thattransport radiolabeled cysteine inward in an ATP-dependent,uncoupled-independent manner. New pleiotropic cydD phenotypes have beenreported, including ones with sensitivity to benzylpenicillin anddithiothreitol, and ones with loss of motility. Both of these phenotypesare consistent with periplasmic defects in disulfide bond formation. Thepresence of exogenous cysteine was able to reverse these phenotypes andaffect the levels of periplasmic c-type cytochromes in cydD andwild-type strains, but did not restore cytochrome d. Consistent withCydDC being a cysteine exporter, cydD mutant growth was hypersensitiveto high cysteine concentrations and produced higher cytoplasmic cysteinelevels, as did a mutant defective in ORF299 which encoded a transporterof the major facilitator superfamily. A cydD ORF299 double mutant wasextremely cysteine-sensitive and had higher cytoplasmic cysteine levels,whereas CydDC overexpression conferred resistance to high extracellularcysteine concentrations. It seems likely that CydDC is responsible forthe export of cysteine, which is crucial for redox homeostasis in theperiplasm (Pittman M. S. et al., J Biol. Chem.; 277(51):49841-9 (2002)).

In addition to YdeD and YfiK, which have been previously reported asL-cysteine exporter proteins in E. coli, the effects of 33 putative drugtransporter genes in E. coli on L-cysteine export and overproduction wasanalyzed. Overexpression of the acrD, acrEF, bcr, cusA, emrAB, emrKY,ybjYZ, and yojlH genes reversed the growth inhibition of tnaA-disruptedE. coli cells by L-cysteine. The tnaA gene is the major cysteinedesulfhydrase gene. It was found that overexpression of these eightgenes reduces intracellular L-cysteine levels after cultivation in thepresence of L-cysteine. Amino acid transport assays showed that Bcroverexpression, which confers bicyclomycin and tetracycline resistance,specifically promotes L-cysteine export driven by the energy generatedfrom the proton gradient. When a tnaA-disrupted E. coli strainexpressing the altered cysE gene was transformed with a plasmid carryingthe bcr gene, the transformant produced more L-cysteine than cellscarrying the vector only. A reporter gene assay suggested that the bcrgene is constitutively expressed at substantial levels. These resultsindicate that the multidrug transporter Bcr in the major facilitatorfamily is involved in L-cysteine export and overproduction ingenetically engineered E. coli cells (Yamada S. et al., Appl EnvironMicrobiol.; 72(7):4735-42 (2006)).

But currently, there have been no reports of using a bacterium having aprotein derived from bacteria belonging to the genus Pantoea, and whichis able to confer resistance to growth inhibition by L-cysteine in thebacterium, for the purpose of producing L-amino acids.

SUMMARY OF THE INVENTION

Aspects of the presently disclosed subject matter can include enhancingthe productivity of L-amino acid-producing strains and providing amethod for producing non-aromatic or aromatic L-amino acids using thesestrains.

The above aspects were achieved by finding that a protein activity whichconfers to a bacterium resistance to growth inhibition by L-cysteine canresult in enhancing production of L-amino acids, such as L-threonine,L-lysine, L-cysteine, L-methionine, L-leucine, L-isoleucine, L-valine,L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-asparticacid, L-glutamine, L-glutamic acid, L-proline, L-arginine,L-phenylalanine, L-tyrosine, and L-tryptophan.

Another aspect of the present invention includes a bacterium of theEnterobacteriaceae family having an increased ability to produce aminoacids, such as L-threonine, L-lysine, L-cysteine, L-methionine,L-leucine, L-isoleucine, L-valine, L-histidine, glycine, L-serine,L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid,L-proline, L-arginine, L-phenylalanine, L-tyrosine, and L-tryptophan.

It is an aspect of the present invention to provide a method forproducing an L-amino acid comprising cultivating an Enterobacteriaceaebacterium that is able to produce an L-amino acid in a culture medium,and collecting the L-amino acid from the culture medium or thebacterium, wherein the bacterium has been modified to increase anactivity of a protein which is able to confer to the bacteriumresistance to growth inhibition by L-cysteine, wherein said protein isselected from the group consisting of:

(A) the protein of SEQ ID NO: 2, or a variant thereof, and

(B) the protein of SEQ ID NO: 4, or a variant thereof.

It is a further aspect of the present invention to provide the method asdescribed above, wherein expression of a DNA encoding said protein insaid bacterium is enhanced.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said bacterium is transformed with a DNAencoding said protein.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the DNA comprises a gene selected from thegroup consisting of c0011 and d0663.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said protein is at least protein (B) or avariant thereof, and said bacterium has been further modified toincrease expression of a DNA encoding the protein of SEQ ID NO: 6; or avariant thereof.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the DNA is the c09478 gene.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium belongs to the genus Escherichia.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium belongs to the genus Pantoea.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium is Escherichia coli.

It is a further aspect of the present invention to provide the method asdescribed above, wherein the bacterium is Pantoea ananatis.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said L-amino acid is selected from the groupconsisting of an aromatic L-amino acid and a non-aromatic L-amino acid.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said aromatic L-amino acid is selected from thegroup consisting of L-phenylalanine, L-tyrosine, and L-tryptophan.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said non-aromatic L-amino acid is selected fromthe group consisting of L-threonine, L-lysine, L-cysteine and L-cysteinederivatives, L-methionine, L-leucine, L-isoleucine, L-valine,L-histidine, glycine, L-serine, L-alanine, L-asparagine, L-asparticacid, L-glutamine, L-glutamic acid, L-proline, L-arginine andO-acetyl-L-serine.

It is a further aspect of the present invention to provide the method asdescribed above, wherein said L-amino acid is selected from the groupconsisting of L-cysteine, L-valine, L-leucine, L-Isoleucine,L-threonine, L-glutamic acid, L-glycine, L-alanine, L-histidine, andO-acetyl-L-serine.

Methods embodying principles of the present invention are described indetail below.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIGS. 1A and 1B show growth curves of strains carrying the plasmidspSTV-c0011 PF and pSTV-PA36 ccd in a medium containing cysteine.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

1. Bacterium

The bacterium can be an L-amino acid-producing bacterium of theEnterobacteriaceae family, wherein the bacterium has a protein derivedfrom bacteria belonging to the genus Pantoea, and the protein is able toconfer resistance to growth inhibition by L-cysteine.

The phrase “L-amino acid-producing bacterium” can mean a bacterium whichhas an ability to produce and excrete an L-amino acid into a medium,when the bacterium is cultured in the medium.

The phrase “L-amino acid-producing bacterium” can also mean a bacteriumwhich is able to produce and cause accumulation of an L-amino acid in aculture medium in an amount larger than a wild-type or parental strainof E. coli, such as E. coli K-12, and preferably can mean that themicroorganism is able to cause accumulation in a medium of an amount notless than 0.5 g/L, and in another embodiment not less than 1.0 g/L, ofthe target L-amino acid. The term “L-amino acid” can include L-alanine,L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid,L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, L-valine, and O-acetyl-L-serine.

The term “aromatic L-amino acid” can include L-phenylalanine,L-tyrosine, and L-tryptophan. The term “non-aromatic L-amino acid” caninclude L-threonine, L-lysine, L-cysteine and L-cysteine derivatives,L-methionine, L-leucine, L-isoleucine, L-valine, L-histidine, glycine,L-serine, L-alanine, L-asparagine, L-aspartic acid, L-glutamine,L-glutamic acid, L-proline, L-arginine and O-acetyl-L-serine.L-threonine, L-lysine, L-cysteine, L-leucine, L-histidine, L-glutamicacid, L-phenylalanine, L-tryptophan, L-proline, L-arginine andO-acetyl-L-serine are other embodiments.

Some of the L-cysteine produced by the bacterium may change intoL-cystine in the medium by the formation of a disulfide bond.S-sulfocysteine may be generated by the reaction of L-cysteine andthiosulfuric acid, which are both present in the medium (Szczepkowski T.W., Nature, vol. 182 (1958)). When S-sulfocysteine is produced in themedium, it can be converted into L-cysteine by reduction with a reducingagent such as dithiothreitol. Furthermore, the L-cysteine that isgenerated in the bacterial cells may be condensed with a ketone,aldehyde, or, for example, pyruvic acid, which is also present in thecells, to produce a thiazolidine derivative via the intermediatehemithioketal (refer to Japanese Patent No. 2992010). The thiazolidinederivative and hemithioketal may exist as an equilibrated mixture. Whena thiazolidine derivative of L-cysteine is produced in the medium,L-cysteine can be produced by collecting the thiazolidine derivativefrom the medium to break the reaction equilibrium between thethiazolidine derivative and L-cysteine so that L-cysteine is produced inexcess. Therefore, the L-cysteine-producing ability is not limited tothe ability to accumulate only L-cysteine in the medium or cells, butalso includes the ability to accumulate L-cystine or derivatives thereofsuch as S-sulfocysteine, a thiazolidine derivative, a hemithioketal, ora mixture thereof in the medium.

Some of the L-cysteine derivatives, such as γ-glutamylcysteine,glutathione, cystathionine, homocysteine, methionine, andS-adenosylmethionine, for example, can be biosynthesized from cysteineas an important starting material. The L-cysteine derivatives also caninclude methylcysteine, ethylcysteine, carbocysteine, sulfocysteine,acetylcysteine, and so forth. L-cysteine obtained as described above canbe used to produce these L-cysteine derivatives.

Fermentative production of these compounds can be achieved by using thecorresponding producer microorganisms as host strains with combinationsof ability to over-produce cysteine. Therefore, the L-cysteine-producingability includes the above compounds, such as γ-glutamylcysteine,glutathione, cystathionine, homocysteine, methionine, andS-denosylmethionine, which produce cysteine as an importantintermediate.

To impart the ability to produce the compounds biosynthesized fromcysteine such as γ-glutamylcysteine, glutathione, cystathionine,homocysteine, methionine and S-adenosylmethionine, methodsconventionally employed in the breeding of coryneform bacteria orbacteria of the genus Escherichia (see “Amino Acid Fermentation”, GakkaiShuppan Center (Ltd.), 1st Edition, published May 30, 1986, pp. 77-100)can be used. Such methods include producing a microorganism having theproperties of an auxotrophic mutant, an analogue-resistant strain, or ametabolic regulation mutant, or by constructing a recombinant strain sothat it overexpresses a corresponding biosynthesis enzyme. Decreasingactivities of enzymes that catalyze reactions which branch off the mainpathway, and/or are involved in degradation of a corresponding compoundor its intermediates, can also be effective to increase the product.Here, in the breeding of each producing bacteria, one or more of theabove described properties may be imparted. The expression ofcorresponding biosynthesis enzyme(s) can be enhanced alone or incombinations of two or more. Furthermore, imparting properties such asan auxotrophic mutation, analogue resistance, or metabolic regulationmutation may be combined with the methods of enhancing the biosynthesisenzymes.

The phrase “bacterium which has a resistance to growth inhibition byL-cysteine” can mean a bacterium derived from a strain of bacterium asthe parent strain, and which has genetic properties so that it can growin a medium containing L-cysteine. A solid medium can be used.

The bacterium which is resistant to growth inhibition by L-cysteineshows better favorable growth as compared with the parent strain whencultured in a medium containing L-cysteine. For example, a bacteriumwhich can form colonies within 20 hours of cultivation at 34° C. onplates with M9 minimal medium containing 50 μM or more, or in anotherexample 200 μM of L-cysteine, can be resistant to L-cysteine.

The strains carrying the genes as described above which can grow fasterthan the control strain in a medium containing 50 μM L-cysteine, or inanother example 200 μM, indicates that this gene or gene locus canconfer cysteine resistance.

The Enterobacteriaceae family includes bacteria belonging to the generaEscherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus,Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, etc.Specifically, those classified into the Enterobacteriaceae according tothe taxonomy used by the NCBI (National Center for BiotechnologyInformation) database(www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) can be used.A bacterium belonging to the genus Escherichia or Pantoea can be used.

The phrase “a bacterium belonging to the genus Escherichia” can meanthat the bacterium is classified into the genus Escherichia according tothe classification known to a person skilled in the art of microbiology.Examples of a bacterium belonging to the genus Escherichia can include,but are not limited to, Escherichia coli (E. coli).

The bacterium belonging to the genus Escherichia is not particularlylimited, however for example, bacteria described by Neidhardt, F. C. etal. (Escherichia coli and Salmonella typhimurium, American Society forMicrobiology, Washington D.C., 1208, Table 1) can be used.

The phrase “a bacterium belonging to the genus Pantoea” can mean thatthe bacterium is classified as the genus Pantoea according to theclassification known to a person skilled in the art of microbiology.Some species of Enterobacter agglomerans have been recentlyre-classified into Pantoea agglomerans, Pantoea ananatis, Pantoeastewartii or the like, based on nucleotide sequence analysis of 16SrRNA, etc (International Journal of Systematic Bacteriology, July 1989,39(3). p. 337-345). Furthermore, some bacteria belonging to the genusErwinia were re-classified as Pantoea ananatis or Pantoea stewartii(International Journal of Systematic Bacteriology, January 1993, 43(1),pp. 162-173). Typical strains of the Pantoea bacteria include, but arenot limited to, Pantoea ananatis, Pantoea stewartii, Pantoeaagglomerans, and Pantoea citrea. Specific examples include the followingstrains: Pantoea ananatis AJ13355 (FERM BP-6614, European PatentPublication No. 0952221), Pantoea ananatis AJ13356 (FERM BP-6615,European Patent Publication No. 0952221), Pantoea ananatis AJ 13601(FERM BP-7207, European Patent Publication No. 0952221), Pantoeaananatis SC17 (FERM BP-11091, European Patent Publication No. 0952221).An exemplary k-Red resistant strain is Pantoea ananatis SC17(0) (VKPMB-9246, RU application 2006134574). The SC17 strain was deposited at theNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depository (address: Tsukuba Central 6,1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Feb.4, 2009 under the Budapest Treaty, and assigned an accession number ofFERM BP-11091. The SC17(0) strain was deposited at the Russian NationalCollection of Industrial Microorganisms (VKPM), GNII Genetika (address:Russia, 117545 Moscow, 1 Dorozhny proezd. 1) on Sep. 21, 2005 with anaccession number of VKPM B-9246, and then converted to internationaldeposit under the Budapest Treaty on Oct. 13, 2006.

According to the presently disclosed subject matter, a bacterium ofEnterobacteriaceae family is modified to increase an activity of theprotein having the amino acid sequence of SEQ ID NO: 2 or its variant,or the protein having the amino acid sequence of SEQ ID NO: 4, or itsvariant, or activities of both of these proteins.

In an embodiment of the presently disclosed subject matter, when thebacterium has been modified to increase at least the protein having theamino acid sequence of SEQ ID NO: 4, or its variant, the bacterium isfurther modified to increase expression of a DNA encoding the protein ofSEQ ID NO: 6, or its variant.

Examples of the protein which has the amino acid sequence of SEQ ID NO:2 or its variant include the c0011 gene. Examples of the protein whichhas the amino acid sequence of SEQ ID NO: 4 or its variant include thec0663 gene. Examples of the protein which has the amino acid sequence ofSEQ ID NO: 6 or its variant include the c09478 gene.

The nucleotide sequence of the c0011 gene from the strain P. ananatisSC17 and the amino acid sequence of protein encoded by the c0011 geneare shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The nucleotide sequence of the d0663 gene from the strain P. ananatisSC17 and the amino acid sequence of protein encoded by the d0663 geneare shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The nucleotide sequence of the c09478 gene from the strain P. ananatisSC17 and the amino acid sequence of protein encoded by the c09478 geneare shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

Since there may be some differences in DNA sequences between the generaor strains of the Enterobacteriaceae family, the c0011, d0663 and c09478genes are not limited to the genes shown in SEQ ID No:1, SEQ ID No:3 andSEQ ID No:5 but may include genes homologous to SEQ ID No:1, SEQ ID No:3and SEQ ID No:5.

The c0011 gene may encode the putative transmembrane protein ofKlebsiella pneumoniae (GenBank Accession No. ABR77369.1), hypotheticalprotein KCO_(—)01469 of Citrobacter koseri (GenBank Accession No.ABV12602.1), Permease (DMT) superfamily of Acinetobacter baumannii(GenBank Accession No. ABO12139.2), and putative membrane protein ofPseudomonas aeruginosa (GenBank Accession No. ABR86455.1).

The d0663 gene may encode the Lysine exporter protein (LYSE/YGGA) ofErwinia tasmaniensis (GenBank Accession No. CA095150.1), putativemembrane protein of Pectobacterium atrosepticum (GenBank Accession No.CAG76207.1), probable transport protein of Chromobacterium violaceum(GenBank Accession No. AAQ59575.1), and Lysine exporter protein(LYSE/YGGA) of Burkholderia vietnamiensis (GenBank Accession No.ABO58107.1).

Therefore, the protein variant encoded by the c0011, d0663, and c09478genes can have a homology of not less than 80%, in another example notless than 90%, in another example not less than 95%, or in anotherexample not less than 98%, and in another example not less than 99%,with respect to the entire amino acid sequences shown in SEQ ID No:2,SEQ ID No:4, and SEQ ID No:6, respectively, as long as the proteinconfers resistance to cysteine. The phrase “protein variant” can meanproteins which have changes in their sequences, whether these changesare deletions, insertions, additions, or substitutions of amino acids.The number of changes in the variant proteins depends on the position inthe three dimensional structure of the protein or the type of amino acidresidues. It may be 1 to 30, in another example 1 to 15, and in anotherexample 1 to 5 in SEQ ID No:2, SEQ ID No:4 and SEQ ID No:6. Thesechanges in the variants can occur in regions of the protein which arenot critical for the three dimensional structure of the protein. This isbecause some amino acids have high homology to one another so the threedimensional structure is not affected by such a change.

Homology between two amino acid sequences can be determined usingwell-known methods, for example, the computer program BLAST 2.0, whichcalculates three parameters: score, identity and similarity. In thisspecification, “homology” can mean “identity”.

The substitution, deletion, insertion, or addition of one or severalamino acid residues can be conservative mutation(s) so that the activityis maintained. A representative conservative mutation can be aconservative substitution. Examples of conservative substitutionsinclude substitution of Ser or Thr for Ala, substitution of Gln, His orLys for Arg, substitution of Glu, Gln, Lys, His or Asp for Asn,substitution of Asn, Glu or Gln for Asp, substitution of Ser or Ala forCys, substitution of Asn, Glu, Lys, His, Asp or Arg for Gln,substitution of Asn, Gln, Lys or Asp for Glu, substitution of Pro forGly, substitution of Asn, Lys, Gln, Arg or Tyr for His, substitution ofLeu, Met, Val or Phe for Be, substitution of Be, Met, Val or Phe forLeu, substitution of Asn, Glu, Gln, His or Arg for Lys, substitution ofBe, Leu, Val or Phe for Met, substitution of Trp, Tyr, Met, Ile or Leufor Phe, substitution of Thr or Ala for Ser, substitution of Ser or Alafor Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe orTrp for Tyr, and substitution of Met, Ile or Leu for Val.

Therefore, the c0011, d0663 and c09478 genes may be variants whichhybridize under stringent conditions with the nucleotide sequences shownin SEQ ID No:1, SEQ ID No:3 and SEQ ID No:5, respectively, or probeswhich can be prepared from these nucleotide sequences, provided that afunctional protein is encoded. “Stringent conditions” include thoseunder which a specific hybrid, for example, a hybrid having homology ofnot less than 60%, and in another example not less than 70%, and inanother example not less than 80%, and in another example not less than90%, and in another example not less than 95%, and in another examplenot less than 98%, and in yet another example not less than 99%, isformed and a non-specific hybrid, for example, a hybrid having homologylower than the above, is not formed. For example, stringent conditionsare exemplified by washing one time or more, and in another example twoor three times, at a salt concentration of 1×SSC, 0.1% SDS, and inanother example 0.1×SSC, 0.1%, at 60° C. Duration of washing depends onthe type of membrane used for blotting and, as a rule, should be what isrecommended by the manufacturer. For example, the recommended durationof washing for the Hybond™ N+ nylon membrane (Amersham) under stringentconditions is 15 minutes. Washing can be performed 2 to 3 times. Thelength of the probe may be suitably selected depending on thehybridization conditions, and can be 100 by to 1 kbp.

The phrase “a bacterium has been modified to increase an activity of aprotein” means that the activity of the protein in the cell is higher ascompared to the non-modified microorganism, for example, a parental orwild-type strain. The activity of the protein can be increased in thecell by enhancing the expression of the gene encoding the protein.Examples of such modification can include increasing the copy number ofexpressed gene per cell, increasing the expression level of the gene,and so forth. The quantity of the copy number of an expressed gene canbe measured, for example, by restricting the chromosomal DNA followed bySouthern blotting using a probe based on the gene sequence, fluorescencein situ hybridization (FISH), and the like. The level of gene expressioncan be measured by various known methods including Northern blotting,quantitative RT-PCR, and the like.

More concretely, enhancing the expression of the c0011, d0663 or c09478gene can be attained by increasing the copy number of the c0011, d0663or c09478 gene, modifying an expression regulatory sequence of thec0011, d0663 or c09478 gene, amplifying a gene encoding a regulatoryfactor that is responsible for increasing expression of the c0011, d0663or c09478 genes, respectively, or disrupting or attenuating a geneencoding a regulatory factor that is responsible for reducing expressionof the c0011, d0663 or c09478 gene, respectively, by using atransformation or a homologous recombination technique.

For example, a recombinant DNA can be prepared by ligating a genefragment containing the c0011, d0663 or c09478 gene to a vector,preferably a multi-copy vector, which can replicate in the hostmicroorganism, and introducing the resulting vector into the hostmicroorganism.

The copy number of the c0011, d0663 or c09478 gene can also be increasedby integrating multiple copies of the gene into a chromosomal DNA of amicroorganism. In order to integrate multiple copies of the c0011, d0663or c09478 gene into a chromosomal DNA of a microorganism, homologousrecombination can be performed by targeting a sequence which exists inmultiple copies on a chromosomal DNA. Repetitive DNA and invertedrepeats at the end of a transposon can be used. Alternatively, asdisclosed in JP2-109985A, it is also possible to incorporate the c0011,d0663 or c09478 gene into a transposon, and allow it to be transferredso that multiple copies of the gene are integrated into the chromosomalDNA. Integration of the c0011, d0663 or c09478 gene into the chromosomecan be confirmed by southern hybridization using a probe having apartial sequence of the c0011, d0663 or c09478 genes.

Enhancing expression of the c0011, d0663 or c09478 gene can also beattained by replacing an expression regulatory sequence, including apromoter of the c0011, d0663 or c09478 gene, on a chromosomal DNA or ona plasmid, with a stronger one, as described in WO 00/18935. Forexample, the lac promoter, trp promoter, trc promoter, P_(L) promoter,and so forth are known as strong promoters. Moreover, it is alsopossible to introduce several nucleotide substitutions into a promoterregion for the c0011, d0663 or c09478 gene so that the promoter isstronger. A method for evaluating the strength of promoters and examplesof strong promoters are disclosed in Goldstein et al. (Prokaryoticpromoters in biotechnology. Biotechnol. Annu. Rev., 1995, 1, 105-128).Furthermore, it is known that a spacer sequence between the ribosomebinding site (RBS) and translation initiation codon, especially, severalnucleotides just upstream of the initiation codon, has a great influenceon translation efficiency. Therefore, this sequence may be modified.Expression regulatory sequences of the c0011, d0663 and c09478 genes maybe identified using a vector for promoter identification or geneticanalysis software such as GENETYX. Expression can also be improved byprolonging the lifetime of the mRNA. Furthermore, enzyme activity canalso be increased by preventing degradation of the enzyme protein.

In order to enhance an activity of the protein encoded by the c0011,d0663 or c09478 gene, a mutation which increases an L-amino acid-exportability may be introduced into the c0011, d0663 or c09478 gene. Examplesof mutations that increase activity of the protein encoded by the c0011,d0663 or c09478 gene (C0011, D0663 or C09478 protein) include a promotersequence mutation that increases the transcription of the c0011, d0663or c09478 genes, and a c0011, d0663 or c09478 gene coding regionmutation that increases the specific activity of the C0011, D0663 orC09478 protein.

Methods for preparation of plasmid DNA, digestion and ligation of DNA,transformation, selection of an oligonucleotide as a primer, and thelike may be typical methods well-known to one skilled in the art. Thesemethods are described, for instance, in Sambrook, J., Fritsch, E. F.,and Maniatis, T., “Molecular Cloning: A Laboratory Manual, SecondEdition”, Cold Spring Harbor Laboratory Press (1989).

L-Amino Acid-Producing Bacteria

Bacteria which are able to produce either an aromatic or a non-aromaticL-amino acids may be used.

The bacterium can be obtained by introducing a gene which encodes aprotein able to confer resistance to cysteine in a bacterium whichinherently has the ability to produce L-amino acids. Alternatively, thebacterium can be obtained by imparting the ability to produce L-aminoacids to a bacterium that already has a protein able to conferresistance to cysteine.

L-Threonine-Producing Bacteria

Examples of parent strains which can be used to deriveL-threonine-producing bacteria include, but are not limited to, strainsbelonging to the genus Escherichia, such as E. coli TDH-6/pVIC40 (VKPMB-3996) (U.S. Pat. No. 5,175,107, U.S. Pat. No. 5,705,371), E. coli472T23/pYN7 (ATCC 98081) (U.S. Pat. No. 5,631,157), E. coli NRRL-21593(U.S. Pat. No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat. No.5,474,918), E. coli FERM BP-3519 and FERM BP-3520 (U.S. Pat. No.5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14,947-956 (1978)), E. coli VL643 and VL2055 (EP 1149911 A), and the like.

The strain TDH-6 is deficient in the thrC gene, as well as beingsucrose-assimilative, and the ilvA gene has a leaky mutation. Thisstrain also has a mutation in the rhtA gene, which imparts resistance tohigh concentrations of threonine or homoserine. The strain B-3996contains the plasmid pVIC40 which was obtained by inserting a thrA*BCoperon which includes a mutant thrA gene into a RSF1010-derived vector.This mutant thrA gene encodes aspartokinase homoserine dehydrogenase Iwhich has substantially desensitized feedback inhibition by threonine.The strain B-3996 was deposited on Nov. 19, 1987 in the All-UnionScientific Center of Antibiotics (Russia, 117105 Moscow, NagatinskayaStreet 3-A) under the accession number RIA 1867. The strain was alsodeposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russia, 117545 Moscow, 1^(st) Dorozhny proezd, 1)on Apr. 7, 1987 under the accession number B-3996.

E. coli VKPM B-5318 (EP 0593792B) may also be used as a parent strain toderive L-threonine-producing bacteria of the present invention. Thestrain B-5318 is prototrophic with regard to isoleucine and atemperature-sensitive lambda-phage C1 repressor and PR promoter replacesthe regulatory region of the threonine operon in plasmid pVIC40. Thestrain VKPM B-5318 was deposited in the Russian National Collection ofIndustrial Microorganisms (VKPM) on May 3, 1990 under accession numberof VKPM B-5318.

The bacterium can be additionally modified to enhance expression of oneor more of the following genes:

-   -   the mutant thrA gene which codes for aspartokinase homoserine        dehydrogenase I resistant to feed back inhibition by threonine;    -   the thrB gene which codes for homoserine kinase;    -   the thrC gene which codes for threonine synthase;    -   the rhtA gene which codes for a putative transmembrane protein;    -   the asd gene which codes for aspartate-β-semialdehyde        dehydrogenase; and    -   the aspC gene which codes for aspartate aminotransferase        (aspartate transaminase);

The thrA gene which encodes aspartokinase homoserine dehydrogenase I ofEscherichia coli has been elucidated (nucleotide positions 337 to 2799,GenBank accession no. NC_(—)000913.2, gi: 49175990). The thrA gene islocated between the thrL and thrB genes on the chromosome of E. coliK-12. The thrB gene which encodes homoserine kinase of Escherichia colihas been elucidated (nucleotide positions 2801 to 3733, GenBankaccession no. NC_(—)000913.2, gi: 49175990). The thrB gene is locatedbetween the thrA and thrC genes on the chromosome of E. coli K-12. ThethrC gene which encodes threonine synthase of Escherichia coli has beenelucidated (nucleotide positions 3734 to 5020, GenBank accession no.NC_(—)000913.2, gi: 49175990). The thrC gene is located between the thrBgene and the yaaX open reading frame on the chromosome of E. coli K-12.All three genes function as a single threonine operon. To enhanceexpression of the threonine operon, the attenuator region which affectsthe transcription can be removed from the operon (WO 2005/049808, WO2003/097839).

A mutant thrA gene which codes for aspartokinase homoserinedehydrogenase I resistant to feed back inhibition by threonine, as wellas the thrB and thrC genes, can be obtained as one operon from thewell-known plasmid pVIC40 which is present in the threonine producing E.coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in U.S.Pat. No. 5,705,371.

The rhtA gene exists at 18 min on the E. coli chromosome close to theglnHPQ operon, which encodes components of the glutamine transportsystem. The rhtA gene is identical to ORF1 (ybiF gene, nucleotidepositions 764 to 1651, GenBank accession number AAA218541, gi:440181)and located between the pexB and ompX genes. The unit expressing aprotein encoded by the ORF1 has been designated the rhtA gene (rht:resistance to homoserine and threonine). Also, it was revealed that therhtA23 mutation is an A-for-G substitution at position −1 with respectto the ATG start codon (ABSTRACTS of the 17^(th) International Congressof Biochemistry and Molecular Biology in conjugation with Annual Meetingof the American Society for Biochemistry and Molecular Biology, SanFrancisco, Calif. Aug. 24-29, 1997, abstract No. 457, EP 1013765 A).

The asd gene of E. coli has already been elucidated (nucleotidepositions 3572511 to 3571408, GenBank accession no. NC_(—)000913.1,gi:16131307), and can be obtained by PCR (polymerase chain reaction;refer to White, T. J. et al., Trends Genet., 5, 185 (1989)) utilizingprimers prepared based on the nucleotide sequence of the gene. The asdgenes of other microorganisms can be obtained in a similar manner.

Also, the aspC gene of E. coli has already been elucidated (nucleotidepositions 983742 to 984932, GenBank accession no. NC_(—)000913.1,gi:16128895), and can be obtained by PCR. The aspC genes of othermicroorganisms can be obtained in a similar manner.

L-Lysine-Producing Bacteria

Examples of L-lysine-producing bacteria belonging to the genusEscherichia include mutants having resistance to an L-lysine analogue.The L-lysine analogue inhibits growth of bacteria belonging to the genusEscherichia, but this inhibition is fully or partially desensitized whenL-lysine is present in a medium. Examples of the L-lysine analogueinclude, but are not limited to, oxalysine, lysine hydroxamate,S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam,and so forth. Mutants having resistance to these lysine analogues can beobtained by subjecting bacteria belonging to the genus Escherichia to aconventional artificial mutagenesis treatment. Specific examples ofbacterial strains useful for producing L-lysine include Escherichia coliAJ11442 (FERM BP-1543, NRRL B-12185; see U.S. Pat. No. 4,346,170) andEscherichia coli VL611. In these microorganisms, feedback inhibition ofaspartokinase by L-lysine is desensitized.

The strain WC196 may be used as an L-lysine producing bacterium ofEscherichia coli. This bacterial strain was bred by conferring AECresistance to the strain W3110, which was derived from Escherichia coliK-12. The resulting strain was designated Escherichia coli AJ13069strain and was deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (currentlyNational Institute of Advanced Industrial Science and Technology,International Patent Organism Depositary, Tsukuba Central 6, 1-1,Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Dec. 6,1994 and received an accession number of FERM P-14690. Then, it wasconverted to an international deposit under the provisions of theBudapest Treaty on Sep. 29, 1995, and received an accession number ofFERM BP-5252 (U.S. Pat. No. 5,827,698).

Examples of parent strains which can be used to deriveL-lysine-producing bacteria also include strains in which expression ofone or more genes encoding an L-lysine biosynthetic enzyme are enhanced.Examples of such genes include, but are not limited to, genes encodingdihydrodipicolinate synthase (dapA), aspartokinase (lysC),dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase(lysA), diaminopimelate dehydrogenase (ddh) (U.S. Pat. No. 6,040,160),phosphoenolpyruvate carboxylase (ppc), aspartate semialdehydedehydrogenase (asd), and aspartase (aspA) (EP 1253195 A). In addition,the parent strains may have increased expression of the gene involved inenergy efficiency (cyo) (EP 1170376 A), the gene encoding nicotinamidenucleotide transhydrogenase (pntAB) (U.S. Pat. No. 5,830,716), the ybjEgene (WO2005/073390), or combinations thereof.

Examples of parent strains which can be used to deriveL-lysine-producing bacteria also include strains having decreased oreliminated activity of an enzyme that catalyzes a reaction whichgenerates a compound other than L-lysine by branching off from thebiosynthetic pathway of L-lysine. Examples of such enzymes includehomoserine dehydrogenase, lysine decarboxylase (U.S. Pat. No.5,827,698), and the malic enzyme (WO 2005/010175).

L-Cysteine-Producing Bacteria

Examples of parent strains which can be used to deriveL-cysteine-producing bacteria include, but are not limited to, strainsbelonging to the genus Escherichia, such as E. coli JM15 which istransformed with different cysE alleles coding for feedback-resistantserine acetyltransferases (U.S. Pat. No. 6,218,168, Russian patentapplication 2003121601); E. coli W3110 having over-expressed genes whichencode proteins suitable for secreting substances toxic for cells (U.S.Pat. No. 5,972,663); E. coli strains having lowered cysteinedesulfhydrase activity (JP11155571A2); E. coli W3110 with increasedactivity of a positive transcriptional regulator for cysteine regulonencoded by the cysB gene (WO0127307A1), and the like.

Bacteria Producing L-Cysteine Derivatives.

Examples of parent strains which can be used to derive bacteriaproducing L-cysteine derivatives include, but are not limited to, thestrains described below. The ability to produce γ-glutamylcysteine canbe achieved, for example, by enhancing activity of γ-glutamylcysteinesynthetase and/or decreasing activity of glutathione synthetase. Theability to produce glutathione can be achieved by enhancing activity ofγ-glutamylcysteine synthetase and/or glutathione synthetase. Usingmutant γ-glutamylcysteine synthetases that are not subject to feedbackinhibition by glutathione can also confer and/or improve ability toproduce glutathione. Methods for microbial production of glutathione aresummarized in Yin et al. (Yin Li, Gongyuan Wei, Jian Chen. ApplMicrobiol Biotechnol (2004) 66: 233-242). The ability to producemethionine can be achieved by conferring L-threonine auxotroph ornorleucine resistance (Japanese Patent Laid-open No. 2000-139471).Inactivation of methionine repressor and/or enhancement of methioninebiosynthetic pathway (i.e., homoserine O-succinyltransferase andcystathionine γ-synthase) can be also effective on conferring andimproving the ability to produce methionine (Japanese Patent Laid-openNo. 2000-139471). Furthermore, using mutant homoserineO-succinyltransferases that are not subject to feed-back inhibition bymethionine can also confer and/or improve the ability to producemethionine. Since cystathionine and homocysteine are intermediates ofmethionine biosynthesis, part of the methods for producing methioninedescribed above can be applied for their production. Specific examplesfor improving cystathionine production are described in Japanese PatentLaid-open No. 2003-010654 (utilization of methionine auxotroph) andJapanese Patent Laid-open No. 2005-16842 (supplement of cysteine orhomoserine in the productive media). Since cystathionine is a precursorof homocysteine, these methods can be applied to homocysteineproduction. The ability to produce S-adenosylmethionine can be achievedby enhancing methionine adenosyltransferase (European Pat. No. 0647712,European Pat. No. 1457569) and/or efflux pump MdfA (U.S. Pat. No.7,410,789).

L-Leucine-Producing Bacteria

Examples of parent strains which can be used to deriveL-leucine-producing bacteria include, but are not limited to, strainsbelonging to the genus Escherichia, such as E. coli strains resistant toleucine (for example, the strain 57 (VKPM B-7386, U.S. Pat. No.6,124,121)) or leucine analogs including β-2-thienylalanine,3-hydroxyleucine, 4-azaleucine, 5,5,5-trifluoroleucine (JP 62-34397 Band JP 8-70879 A); E. coli strains obtained by the gene engineeringmethod described in WO 96/06926; E. coli H-9068 (JP 8-70879 A), and thelike.

The bacterium may be improved by enhancing the expression of one or moregenes involved in L-leucine biosynthesis. Examples include genes of theleuABCD operon, which are preferably represented by a mutant leuA genecoding for isopropylmalate synthase which is not subject to feedbackinhibition by L-leucine (U.S. Pat. No. 6,403,342). In addition, thebacterium may be improved by enhancing the expression of one or moregenes coding for proteins which are responsible for secretion of L-aminoacids from the bacterial cell. Examples of such genes include the b2682and b2683 genes (ygaZH genes) (EP 1239041 A2).

L-Histidine-Producing Bacteria

Examples of parent strains which can be used to deriveL-histidine-producing bacteria include, but are not limited to, strainsbelonging to the genus Escherichia, such as E. coli strain 24 (VKPMB-5945, RU2003677); E. coli strain 80 (VKPM B-7270, RU2119536); E. coliNRRL B-12116-B12121 (U.S. Pat. No. 4,388,405); E. coli H-9342 (FERMBP-6675) and H-9343 (FERM BP-6676) (U.S. Pat. No. 6,344,347); E. coliH-9341 (FERM BP-6674) (EP1085087); E. coli AI80/pFM201 (U.S. Pat. No.6,258,554) and the like.

Examples of parent strains which can be used to deriveL-histidine-producing bacteria also include strains in which expressionof one or more genes encoding an L-histidine biosynthetic enzyme areenhanced. Examples of such genes include genes encoding ATPphosphoribosyltransferase (hisG), phosphoribosyl AMP cyclohydrolase(hist), phosphoribosyl-ATP pyrophosphohydrolase (hisIE),phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase(hisA), amidotransferase (hisH), histidinol phosphate aminotransferase(hisC), histidinol phosphatase (hisB), histidinol dehydrogenase (hisD),and so forth.

It is known that the L-histidine biosynthetic enzymes encoded by hisGand hisBHAFI are inhibited by L-histidine, and therefore anL-histidine-producing ability can also be efficiently enhanced byintroducing a mutation into ATP phosphoribosyltransferase which impartsresistance to the feedback inhibition (Russian Patent Nos. 2003677 and2119536).

Specific examples of strains having an L-histidine-producing abilityinclude E. coli FERM-P 5038 and 5048 which have been transformed by avector carrying a DNA encoding an L-histidine-biosynthetic enzyme (JP56-005099 A), E. coli strains transformed with rht, a gene for an aminoacid-export (EP1016710A), E. coli 80 strain imparted withsulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance(VKPM B-7270, Russian Patent No. 2119536), and so forth.

L-Glutamic Acid-Producing Bacteria

Examples of parent strains which can be used to derive L-glutamicacid-producing bacteria include, but are not limited to, strainsbelonging to the genus Escherichia, such as E. coli VL334thrC⁺ (EP1172433). E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonineauxotrophic strain having mutations in thrC and ilvA genes (U.S. Pat.No. 4,278,765). A wild-type allele of the thrC gene was transferred bythe method of general transduction using a bacteriophage P1 which wasgrown on wild-type E. coli K12 (VKPM B-7) cells. As a result, anL-isoleucine auxotrophic strain VL334thrC⁺ (VKPM B-8961), which is ableto produce L-glutamic acid, was obtained.

Examples of parent strains which can be used to derive L-glutamicacid-producing bacteria include, but are not limited to, strains whichare deficient in α-ketoglutarate dehydrogenase activity, or strains inwhich one or more genes encoding an L-glutamic acid biosynthetic enzymeare enhanced. Examples of the genes involved in L-glutamic acidbiosynthesis include genes encoding glutamate dehydrogenase (gdhA),glutamine synthetase (glnA), glutamate synthetase (gltAB), isocitratedehydrogenase (icdA), aconitate hydratase (acnA, acnB), citrate synthase(gltA), phosphoenolpyruvate carboxylase (ppc), pyruvate carboxylase(pyc), pyruvate dehydrogenase (aceEF, lpdA), pyruvate kinase (pykA,pykF), phosphoenolpyruvate synthase (ppsA), enolase (eno),phosphoglyceromutase (pgmA, pgml), phosphoglycerate kinase (pgk),glyceraldehyde-3-phophate dehydrogenase (gapA), triose phosphateisomerase (tpiA), fructose bisphosphate aldolase (fbp),phosphofructokinase (pfkA, pfkB), and glucose phosphate isomerase (pgi).

Examples of strains which have been modified so that expression of thecitrate synthetase gene, the phosphoenolpyruvate carboxylase gene,and/or the glutamate dehydrogenase gene is/are enhanced include thosedisclosed in EP1078989A, EP955368A, and EP952221A.

Examples of strains which have been modified so that expression of thecitrate synthetase gene and/or the phosphoenolpyruvate carboxylase geneare reduced, and/or are deficient in α-ketoglutarate dehydrogenaseactivity include those disclosed in EP1078989A, EP955368A, andEP952221A.

Examples of parent strains which can be used to derive the L-glutamicacid-producing bacteria also include strains having decreased oreliminated activity of an enzyme that catalyzes synthesis of a compoundother than L-glutamic acid by branching off from an L-glutamic acidbiosynthesis pathway. Examples of such enzymes include isocitrate lyase(aceA), α-ketoglutarate dehydrogenase (sucA), phosphotransacetylase(pta), acetate kinase (ack), acetohydroxy acid synthase (ilvG),acetolactate synthase (ilvI), formate acetyltransferase (pfl), lactatedehydrogenase (ldh), and glutamate decarboxylase (gadAB). Bacteriabelonging to the genus Escherichia deficient in the α-ketoglutaratedehydrogenase activity or having a reduced α-ketoglutarate dehydrogenaseactivity and methods for obtaining them are described in U.S. Pat. Nos.5,378,616 and 5,573,945. Specifically, these strains include thefollowing:

E. coli W3110sucA::Km^(R)

E. coli AJ12624 (FERM BP-3853)

E. coli AJ12628 (FERM BP-3854)

E. coli AJ12949 (FERM BP-4881)

E. coli W3110sucA::Km^(R) is a strain obtained by disrupting theα-ketoglutarate dehydrogenase gene (hereinafter referred to as “sucAgene”) of E. coli W3110. This strain is completely deficient in theα-ketoglutarate dehydrogenase.

Other examples of L-glutamic acid-producing bacterium include thosewhich belong to the genus Escherichia and have resistance to an asparticacid antimetabolite. These strains can also be deficient in theα-ketoglutarate dehydrogenase activity and include, for example, E. coliAJ13199 (FERM BP-5807) (U.S. Pat. No. 5,908,768), FFRM P-12379, whichadditionally has a low L-glutamic acid decomposing ability (U.S. Pat.No. 5,393,671); AJ13138 (FERM BP-5565) (U.S. Pat. No. 6,110,714), andthe like.

Examples of L-glutamic acid-producing bacteria include mutant strainsbelonging to the genus Pantoea which are deficient in theα-ketoglutarate dehydrogenase activity or have a decreasedα-ketoglutarate dehydrogenase activity, and can be obtained as describedabove. Such strains include Pantoea ananatis AJ13356. (U.S. Pat. No.6,331,419). Pantoea ananatis AJ13356 was deposited at the NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry(currently, National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary, Central 6, 1-1,Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on Feb. 19,1998 under an accession number of FERM P-16645. It was then converted toan international deposit under the provisions of Budapest Treaty on Jan.11, 1999 and received an accession number of FERM BP-6615. Pantoeaananatis AJ13356 is deficient in the α-ketoglutarate dehydrogenaseactivity as a result of disruption of the αKGDH-E1 subunit gene (sucA).The above strain was identified as Enterobacter agglomerans when it wasisolated and deposited as the Enterobacter agglomerans AJ13356. However,it was recently re-classified as Pantoea ananatis on the basis ofnucleotide sequencing of 16S rRNA and so forth. Although AJ13356 wasdeposited at the aforementioned depository as Enterobacter agglomerans,for the purposes of this specification, they are described as Pantoeaananatis.

L-Phenylalanine-Producing Bacteria

Examples of parent strains which can be used to deriveL-phenylalanine-producing bacteria include, but are not limited to,strains belonging to the genus Escherichia, such as E. coli AJ12739(tyrA::Tn10, tyrR) (VKPM B-8197); E. coli HW1089 (ATCC 55371) harboringthe mutant pheA34 gene (U.S. Pat. No. 5,354,672); E. coli MWEC101-b(KR8903681); E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRLB-12147 (U.S. Pat. No. 4,407,952). Also, as a parent strain, E. coliK-12 [W3110 (tyrA)/pPHAB (FERM BP-3566), E. coli K-12 [W3110(tyrA)/pPHAD] (FERM BP-12659), E. coli K-12 [W3110 (tyrA)/pPHATerm](FERM BP-12662) and E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] namedas AJ 12604 (FERM BP-3579) may be used (EP 488424 B1). Furthermore,L-phenylalanine producing bacteria belonging to the genus Escherichiawith an enhanced activity of the protein encoded by the yedA gene or theyddG gene may also be used (U.S. patent application publication nos.2003/0148473 A1 and 2003/0157667 A1).

L-Tryptophan-Producing Bacteria

Examples of parent strains which can be used to derive theL-tryptophan-producing bacteria include, but are not limited to, strainsbelonging to the genus Escherichia, such as E. coli JP4735/pMU3028(DSM10122) and JP6015/pMU91 (DSM10123) which is deficient in thetryptophanyl-tRNA synthetase encoded by mutant trpS gene (U.S. Pat. No.5,756,345); E. coli SV164 (pGH5) having a serA allele encodingphosphoglycerate dehydrogenase free from feedback inhibition by serineand a trpE allele encoding anthranilate synthase free from feedbackinhibition by tryptophan (U.S. Pat. No. 6,180,373); E. coli AGX17(pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) deficient inthe enzyme tryptophanase (U.S. Pat. No. 4,371,614); E. coliAGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing abilityis enhanced (WO 97/08333, U.S. Pat. No. 6,319,696), and the like may beused. L-tryptophan-producing bacteria belonging to the genus Escherichiawith an enhanced activity of the identified protein encoded by and theyedA gene or the yddG gene may also be used (U.S. patent applicationpublication nos. 2003/0148473 A1 and 2003/0157667 A1).

Examples of parent strains which can be used to derive theL-tryptophan-producing bacteria also include strains in which one ormore activities of the enzymes selected from anthranilate synthase,phosphoglycerate dehydrogenase, and tryptophan synthase are enhanced.The anthranilate synthase and phosphoglycerate dehydrogenase are bothsubject to feedback inhibition by L-tryptophan and L-serine, so that amutation desensitizing the feedback inhibition may be introduced intothese enzymes. Specific examples of strains having such a mutationinclude a E. coli SV164 which harbors desensitized anthranilate synthaseand a transformant strain obtained by introducing into the E. coli SV164the plasmid pGH5 (WO 94/08031), which contains a mutant serA geneencoding feedback-desensitized phosphoglycerate dehydrogenase.

Examples of parent strains which can be used to derive theL-tryptophan-producing bacteria also include strains into which thetryptophan operon which contains a gene encoding desensitizedanthranilate synthase has been introduced (JP 57-71397 A, JP 62-244382A, U.S. Pat. No. 4,371,614). Moreover, L-tryptophan-producing abilitymay be imparted by enhancing expression of a gene which encodestryptophan synthase, among tryptophan operons (trpBA). The tryptophansynthase includes α and β subunits which are encoded by the trpA andtrpB genes, respectively. In addition, L-tryptophan-producing abilitymay be improved by enhancing expression of the isocitrate lyase-malatesynthase operon (WO 2005/103275).

L-Proline-Producing Bacteria

Examples of parent strains which can be used to deriveL-proline-producing bacteria include, but are not limited to, strainsbelonging to the genus Escherichia, such as E. coli 702ilvA (VKPMB-8012) which is deficient in the ilvA gene and is able to produceL-proline (EP 1172433). The bacterium may be improved by enhancing theexpression of one or more genes involved in L-proline biosynthesis.Examples of such genes for L-proline producing bacteria which arepreferred include the proB gene coding for glutamate kinase of whichfeedback inhibition by L-proline is desensitized (DE Patent 3127361). Inaddition, the bacterium may be improved by enhancing the expression ofone or more genes coding for proteins excreting L-amino acid frombacterial cell. Such genes are exemplified by the b2682 and b2683 genes(ygaZH genes) (EP1239041 A2).

Examples of bacteria belonging to the genus Escherichia, which have anactivity to produce L-proline include the following E. coli strains:NRRL B-12403 and NRRL B-12404 (GB Patent 2075056), VKPM B-8012 (Russianpatent application 2000124295), plasmid mutants described in DE Patent3127361, plasmid mutants described by Bloom F. R. et al (The 15^(th)Miami winter symposium, 1983, p. 34), and the like.

L-Arginine-Producing Bacteria

Examples of parent strains which can be used to deriveL-arginine-producing bacteria include, but are not limited to, strainsbelonging to the genus Escherichia, such as E. coli strain 237 (VKPMB-7925) (U.S. Patent Application Publication No. 2002/058315 A1) and itsderivative strains harboring mutant N-acetylglutamate synthase (RussianPatent Application No. 2001112869), E. coli strain 382 (VKPM B-7926)(EP1170358A1), an arginine-producing strain into which argA geneencoding N-acetylglutamate synthetase is introduced therein(EP1170361A1), and the like.

Examples of parent strains which can be used to derive L-arginineproducing bacteria also include strains in which expression of one ormore genes encoding an L-arginine biosynthetic enzyme are enhanced.Examples of such genes include genes encoding N-acetylglutamyl phosphatereductase (argC), ornithine acetyl transferase (argJ), N-acetylglutamatekinase (argB), acetylornithine transaminase (argD), ornithine carbamoyltransferase (argF), argininosuccinic acid synthetase (argG),argininosuccinic acid lyase (argH), and carbamoyl phosphate synthetase(carAB).

L-Valine-Producing Bacteria

Examples of parent strains which can be used to deriveL-valine-producing bacteria include bacteria belonging to the genusEscherichia such as H-81 (VKPM B-8066), NRRL B-12287 and NRRL B-12288(U.S. Pat. No. 4,391,907), VKPM B-4411 (U.S. Pat. No. 5,658,766), VKPMB-7707 (European patent application EP1016710A2), or the like.

Example of parent strains which can be used to derive L-valine-producingbacteria include, but are not limited to, strains which have beenmodified to overexpress the ilvGMEDA operon (U.S. Pat. No. 5,998,178).It is desirable to remove the region of the ilvGMEDA operon which isrequired for attenuation so that expression of the operon is notattenuated by the L-valine that is produced. Furthermore, the ilvA genein the operon can be disrupted so that threonine deaminase activity isdecreased.

Examples of parent strains which can be used to deriveL-valine-producing bacteria also include mutants having a mutation ofamino-acyl t-RNA synthetase (U.S. Pat. No. 5,658,766). For example, E.coli VL1970, which has a mutation in the ileS gene encoding isoleucinetRNA synthetase, can be used. E. coli VL1970 has been deposited in theRussian National Collection of Industrial Microorganisms (VKPM) (Russia,117545 Moscow, 1^(st) Dorozhny Proezd, 1) on Jun. 24, 1988 underaccession number VKPM B-4411.

Furthermore, mutants requiring lipoic acid for growth and/or lackingH⁺-ATPase can also be used as parent strains (WO 96/06926).

L-Isoleucine-Producing Bacteria

Examples of parent strains which can be used to derive L-isoleucineproducing bacteria include, but are not limited to, mutants havingresistance to 6-dimethylaminopurine (JP 5-304969 A), mutants havingresistance to an isoleucine analogue such as thiaisoleucine andisoleucine hydroxamate, and mutants additionally having resistance toDL-ethionine and/or arginine hydroxamate (JP 5-130882 A). In addition,recombinant strains transformed with genes encoding proteins involved inL-isoleucine biosynthesis, such as threonine deaminase andacetohydroxate synthase, can also be used as parent strains (JP 2-458 A,FR 0356739, and U.S. Pat. No. 5,998,178).

L-Tyrosine-Producing Bacteria

Examples of tyrosine-producing bacteria include Escherichia bacteriawith a desensitized prephenate dehydratase gene (tyrA). The expressionproduct of this gene is desensitized to inhibition by tyrosine (EuropeanPatent Application Laid-open No. 1616940).

2. Method for Producing L-Amino Acid

A method is described for producing an L-amino acid by cultivating abacterium as described herein in a culture medium to produce and excretethe L-amino acid into the medium, and collecting the L-amino acid fromthe medium.

The cultivation, collection, and purification of an L-amino acid fromthe medium and the like may be performed in a manner similar toconventional fermentation methods wherein an amino acid is producedusing a bacterium.

The medium used for culture may be either a synthetic or natural medium,so long as the medium includes a carbon source and a nitrogen source andminerals and, if necessary, appropriate amounts of nutrients which thebacterium requires for growth. As the carbon source, saccharides such asglucose, fructose, sucrose, molasses and starch hydrolysate, and organicacids such as fumaric acid, citric acid and succinic acid, alcohol cuahcas ethanol and glycerol, can be used. As the nitrogen source, variousammonium salts such as ammonia and ammonium sulfate, other nitrogencompounds such as amines, a natural nitrogen source such as peptone,soybean-hydrolysate, and digested fermentative microorganism can beused. As minerals, potassium monophosphate, magnesium sulfate, sodiumchloride, ferrous sulfate, manganese sulfate, calcium chloride, and thelike can be used. As vitamins, thiamine, yeast extract, and the like,can be used.

The cultivation can be performed under aerobic conditions, such as byshaking and/or stirring with aeration, at a temperature of 20 to 40° C.,or in another example 30 to 38° C. The pH of the culture is usuallybetween 5 and 9, or in another example between 6.5 and 7.2. The pH ofthe culture can be adjusted with ammonia, calcium carbonate, variousacids, various bases, and buffers. Usually, a 1 to 5-day cultivationleads to accumulation of the target L-amino acid in the liquid medium.

After cultivation, solids such as cells can be removed from the liquidmedium by centrifugation or membrane filtration, and then the L-aminoacid can be collected and purified by ion-exchange (Nagai, H. et al.,Separation Science and Technology, 39(16), 3691-3710), concentration,membrane separation method (Japanese Patent Laid-open Nos. 9-164323 and9-173792), crystallization methods (WO 2008/078448, WO 2008/078646),and/or other methods.

The L-amino acid collected can contain bacterial cells, mediumcomponents, moisture, and by-product metabolites of the microorganism inaddition to the L-amino acid. Purity of the collected L-amino acid is,for example, 50% or higher, 85% or higher, or even 95% or higher (U.S.Pat. No. 5,431,933, Japanese Patent Publication No. 1-214636, U.S. Pat.Nos. 4,956,471, 4,777,051, 4,946,654, 5,840,358, 6,238,714, U.S. PatentApplication Publication No. 2005/0025878).

L-Cysteine obtained as described above can be used for production ofL-cysteine derivatives. The L-cysteine derivatives includemethylcysteine, ethylcysteine, carbocisteine, sulfocysteine,acetylcysteine, and so forth.

Furthermore, when a thiazolidine derivative of L-cysteine is accumulatedin the medium, L-cysteine can be produced by changing the reactionequilibrium between the thiazolidine derivative and L-cysteine to theL-cysteine side. Furthermore, when S-sulfocysteine is accumulated in themedium, it can be converted into L-cysteine by reduction using areducing agent such as dithiothreitol.

EXAMPLES

The present invention will be more concretely explained below withreference to the following non-limiting examples.

Example 1 Over-Expression of Each of a Gene c0011 and a Gene Locusd0663-c09478

1-1. Cloning of c0011 Gene from P. ananatis SC17.

A DNA fragment containing approximate 300 nt upstream and 200 ntdownstream of c00110RF was obtained by PCR from genomic DNA of P.ananatis SC17 using primers P1 (SEQ ID NO: 7) and P2 (SEQ ID NO: 8) withPrimeSTAR DNA polymerase (Takara Bio Inc.) (5 sec at 98° C., 10 sec at55° C., 4 min at 72° C. for 30 cycles after 5 min at 94° C.).

The obtained 1.4 kb DNA fragment was cloned into a plasmid pSTV29(Takara Bio Inc.) using BamHI sites designed at the 5′ end of eachprimer to obtain the plasmid pSTV-c0011 PF, in which c0011 gene wasoriented in the same direction as the lacZ promoter located on pSTV29.

1-2. Cloning of d0663-c09478 Gene Locus from P. ananatis SC17.

A DNA fragment containing approximate 200 nt downstream of d0663 ORFthrough approximate 200 nt downstream of c09478 ORF was obtained by PCRfrom genomic DNA of P. ananatis SC17 using primers P3 (SEQ ID NO: 9) andP4 (SEQ ID NO: 10) with PrimeSTAR DNA polymerase (Takara Bio Inc.) (5sec at 98° C., 10 sec at 55° C., 3 min at 72° C. for 30 cycles after 5min at 94° C.). The obtained 2.6 kb DNA fragment was cloned into pSTV29using BamHI sites designed at the 5′ end of each primer to obtain theplasmid pSTV-PA36 ccd, in which c09478 gene was oriented in the samedirection as the lacZ promoter located on pSTV29.

1-3. Effect of c0011 and d0663-c09478 on Resistance to Cysteine.

To test the effect of c0011 gene and the gene locus containing two genesd0663 and c09478 on the resistance of cysteine, pSTV-c0011 PF andpSTV-PA36 ccd, and the corresponding vector pSTV29 as a control wereintroduced in E. coli strain MG1655 (ATCC47076, ATCC 700926) and eachtransformant was grown in M9 minimal medium (Sambrook et al., Molecularcloning, 3rd edition, 2001 Cold Spring Harbor Laboratory Press)containing a toxic concentration of L-cysteine for wild-type or controlstrains. Each of the overnight cultures cultivated at 34° C. in a testtube containing 3 ml of M9 minimal medium supplimented with 0.4% glucose(M9Glc media) was diluted 1:100 into fresh M9Glc medium containing 50 μMcysteine in a total of 3 ml and grown at 34° C. in test tubes withagitation overnight. Then the cells were inoculated to 4 ml of freshM9Glc medium in test tubes containing 0 and 200 μM of L-cysteine to givean initial OD₆₆₀ of 0.007. OD₆₆₀ nm was monitored automatically usingTN-1506 incubator (Advantec Toyo, Tokyo, Japan). For maintenance of theplasmids, 25 mg/L of chloramphenicol was supplemented into the medium.The obtained growth curves are shown in FIG. 1. As can be seen in FIG.1, the strains carrying the plasmids pSTV-c0011 PF and pSTV-PA36 ccdgrew faster than the control strain carrying the vector pSTV29 under 200μM cysteine conditions indicating that these gene/gene locus conferresistance to cysteine. The effects can possibly be attributed to theefflux of cysteine by c0011 and d0663 in which the toxic effects ofintercellular cysteine are relieved, since their secondary structurepredictions suggest that they have multiple transmembrane helixes thatare typical of transporters which are able to transport small moleculesacross the membrane.

Example 2 Effect of c0011 on the Fermentative Production of Cysteine

2-1. Construction of a Plasmid for Over-Expression of c0011.

Two types of expression plasmids pMIV-Pnlp0 and pNIV-Pnlp23 withdifferent promoter strength were used for the over-expression of c0011.These plasmids contain the strong nlp0 promoter (or nlp23 promoter) andthe rrnB terminator so that the target genes can be cloned between thesestructures to form an expression unit. The promoter “Pnlp0” (SEQ ID NO:11) was derived from the promoter of the nlpD gene of E. coli K-12,“Pnlp23” (SEQ ID NO: 12) and “Pnlp8” (SEQ ID NO: 13) (see below forfurther details) are the variants with different strengths of thepromoter activity (U.S. Patent Application Publication No. 2010/209977).The overall scheme of the construct of these expression plasmids isdescribed below (see Reference Example 1) as a construct of plasmidpMIV-Pnlp0-YeaS3 where yeaS gene from E. coli K-12 is cloned betweennlp0 promoter and rrnB terminator using the unique restriction sites ofSalI and XbaI designed at the start and end of the ORF respectively. Inorder to obtain pMIV-Pnlp0-based expression plasmid for c0011, the sameconstruct with SalI and XbaI can be applied to replace the yeaS gene onthe pMIV-Pnlp0-YeaS3 with c0011 gene to yield pMIV-Pnlp0-c0011. Also,pMIV-Pnlp23-c0011 can be obtained with the same schematic approach usingSalI and XbaI since the basic structures of these two plasmids aremostly the same except they have a few nucleotides changes in thepromoter region.

A DNA fragment containing c00110RF was obtained by PCR from genomic DNAof P. ananatis SC17 using primers P5 (SEQ ID NO: 14) and P6 (SEQ ID NO:15) with PrimeSTAR DNA polymerase (Takara Bio Inc.) (5 sec at 98° C., 5sec at 55° C., 90 sec at 72° C. for 30 cycles after 5 min at 94° C.).The resulting DNA fragment was cloned into pMIV-Pnlp0 and pMIV-Pnlp23using SalI and XbaI sites designed at the 5′ end of each primer toobtain plasmids pMIV-Pnlp0-c0011 and pMIV-Pnlp23-c0011, respectively.The plasmid pMIV-5JS (JP2008-99668, EP1942183)) was routinely used for acorresponding empty vector as a control.

2-2. Construction of the Cysteine Producing Strains.

To prepare E. coli and P. ananatis strains which are capable offermentative production of cysteine, a plasmid carrying cysEX gene whichencodes a feedback resistant mutant of O-acetyl-L-serine sulfhydrylase(U.S. Patent Application Publication No. 2005/0112731 A1), an essentialfactor for the cysteine production, was constructed. For this purpose,pACYC-DE1, which carries cysEX and ydeD gene (U.S. Pat. No. 5,972,663A),was used as a start material. Construction of pACYC-DE1 is described inU.S. Patent Application Publication No. 2010/209977, as the constructionof pACYC-DES (U.S. Patent Application Publication No. 2005/124049, thisplasmid carries three genes cysEX, ydeD and serA5 (U.S. Pat. No.6,180,373)), in which the cloning step of serA5 on the plasmid isomitted so that only cysEX and ydeD are placed on the plasmid. ThenpACYC-DE1 was digested with restriction enzyme M/uI and was subjected toself-ligation to delete the 330 bp region inside the ORF of ydeD to givepACYC-E1. Thus, the plasmid pACYC-E1 contains only cysEX, whoseintroduction provides the simplest cysteine producing strains. E. coliMG1655 and P. ananatis SC17 strains were transformed with pACYC-E1 toobtain the strains MG1655/pACYC-E1 and SC17/pACYC-E1, respectively,which were used as the basic producer strains for the fermentativeproduction of cysteine.

2-3. Effect of c0011 on the Production of Cysteine in E. coli.

To test the effects of c0011 over-expression on the production ofcysteine, pMIV-Pnlp0-c0011, pMIV-Pnlp23-c0011, and pMIV-5JS (control)were introduced into the cysteine producing strain MG1655/pACYC-E1. Theresulting strains were subjected to the production tests comparingcapacity of the fermentative production of cysteine. Components of themedium for the fermentation experiments are described below.

The composition of the fermentation medium was as follows (final conc.):

Component 1:

(NH₄)₂SO₄ 15 g/L KH₂PO₄ 1.5 g/L MgSO₄•7H₂O   1 g/L thiamine HCl 0.1 mg/L

Component 2:

FeSO₄•7H₂O 1.7 mg/L Na₂MoO₄•2H₂O 0.15 mg/L  CoCl₂•6H₂O 0.7 mg/LMnCl•4H₂O 1.6 mg/L ZnSO₄•7H₂O 0.3 mg/L CuSO₄•5H₂O 0.25 mg/L 

Component 3:

Bacto Tryptone 0.6 g/L Bacto Yeast Extract 0.3 g/L NaCl 0.6 g/L

Component 4:

CaCO₃ 20 g/L

Component 5:

L-histidine HCl H₂O 135 mg/L

Component 6:

Na₂S₂O₃ 4 g/L

Component 7:

pyridoxine HCl 2 mg/L

Component 8:

Glucose 40 g/L

Each component was prepared as a concentrated stock of 10× (component1), 1000× (component 2), 100/6× (component 3), 100× (component 5),350/4× (component 6), 1000× (component 7) and 10× (component 8),respectively. Sterilization conditions were: autoclaving at 110° C. for30 min (component 1, 2, 3, 5 and 8), dry heat sterilization at 180° C.for more than 5 hrs (component 4) and filter sterilization (component 6and 7).

The fermentation was carried out as described in the following. Eachproducing strain (MG1655/pACYC-E1) carrying pMIV-Pnlp0-c0011,pMIV-Pnlp23-c0011, and pMIV-5JS was streaked onto LB plate and grown at34° C. overnight. A loop-full of cells (scratched cells over 7 cm using10 micro litter blue loop (NUNC) from the fully grown plate culture)were taken and inoculated into 2 ml of the medium for fermentation inthe test tubes (23 mm internal diameter; all test tubes were 200 mmlong) to initiate cultivation. The cultivation was carried out at 32° C.with agitation and then terminated when all the sugar was consumed (ittook 21 to 24 hrs depending on the samples). The amount ofcysteine-related compounds which were produced in the culture broth wasdetermined routinely by the method described by Gaitonde, M. K. (BiochemJ.; 104(2):627-33 (1967)). To maintain the plasmids, 25 mg/L ofchloramphenicol and 20 mg/L of tetracycline was supplemented into themedium over the course of cultivation.

The amounts of the cysteine-related compounds and yield against consumedsugar with corresponding values of standard deviation from (fourindependent test tube fermentation) are shown in Table 1. As can be seenfrom Table 1, over-expression of c0011 has positive effects onincreasing the production of cysteine (including cysteine-relatedcompounds) in E. coli, which is likely to be due to its capacity forefflux of cysteine.

TABLE 1 Strain Cysteine (mg/L) Yield (%) MG1655/pACYC-E1/pMIV-5JS 160 ±57 0.40 ± 0.14 MG1655/pACYC-E1/pMIV-Pnlp23-  737 ± 101 1.84 ± 0.25 c0011MG1655/pACYC-E1/pMIV-Pnlp0- 565 ± 83 1.41 ± 0.21 c0011

2-4. Effect of c0011 on the Production of Cysteine in P. ananatis.

To test the effect of c0011 over-expression on the production ofcysteine in P. ananatis, the plasmids pMIV-Pnlp0-c0011,pMIV-Pnlp23-c0011, and pMIV-5JS (control) were introduced into thecysteine-producing strain SC17/pACYC-E1. The productive cultivation wasexecuted as described in the experiment with E. coli except thecultivation time was 18 hrs. Amounts of cysteine-related compounds andyield against consumed sugar with corresponding values of standarddeviation from data for each strain (four independent test tubefermentations) are shown in Table 2. As can be seen from Table 2,over-expression of c0011 has positive effects on increasing theproduction of cysteine in P. ananatis.

TABLE 2 Strain Cysteine (mg/L) Yield (%) SC17/pACYC-E1/pMIV-5JS 95 ± 60.24 ± 0.018 SC17/pACYC-E1/pMIV-Pnlp23-c0011 248 ± 71 0.41 ± 0.012SC17/pACYC-E1/pMIV-Pnlp0-c0011 209 ± 16 0.35 ± 0.027

Example 3 Effect of c0011 on the Fermentative Production of Amino Acids

3-1. Effect of c0011 on the Production of Amino Acids in E. coli.

To test the effect of c0011 over-expression on the production of aminoacids other than cysteine, pMIV-Pnlp0-c0011 and pMIV-5JS (control) wereintroduced into MG1655. Resulting strains were subjected to theproduction tests comparing capacity of the fermentative production ofamino acids. The productive cultivation was executed as described in theexperiment for cysteine production with E. coli, except cultivation timewas 19-24.5 hrs. For maintenance of the plasmids, 25 mg/L ofchloramphenicol was supplemented into the medium during the course ofcultivation. Quantitative analysis of produced L-amino acids in theculture medium was performed using Amino Acid Analyzer L-8900 (HITACHI).Amounts of amino acids with corresponding values of standard deviationfrom data for each strain (four independent test tube fermentations) areshown in Table 3. As can be seen in Table 3, over-expression of c0011has positive effects on increasing the production of valine, leucine,isoleucine, threonine, alanine, glutamate, histidine and glycine in E.coli.

TABLE 3 Val Leu Ile Thr Glu Gly Ala His Strain (mg/L) (mg/L) (mg/L)(mg/L) (mg/L) (mg/L) (mg/L) (mg/L) MG1655/pMIV-5JS   13 ± 2.5 5.2 ± 1.80.4 ± 0.7 2.7 ± 0.9 891 ± 289 0.4 ± 0.3 54 ± 28 14 ± 2.5MG1655/pMIV-Pnlp0- 320 ± 14 205 ± 41  65 ± 11 101 ± 41  1470 ± 15  6.1 ±0.8 307 ± 13  37 ± 3.4 c0011

3-2. Effect of c0011 on the Production of Amino Acids in P. ananatis.

To test the effect of c0011 over-expression on the production of aminoacids other than cysteine, pMIV-Pnlp0-c0011 and pMIV-5JS (control) wereintroduced into SC17. Resulting strains were subjected to the productiontests comparing capacity of the fermentative production of amino acids.The productive cultivation was executed as described in the experimentfor cysteine production with E. coli, except the initial glucoseconcentration was set to 60 g/L and cultivation time was 16 hrs. Formaintenance of the plasmids, 25 mg/L of chloramphenicol was supplementedinto the medium during the course of cultivation. Quantitative analysisof produced amino acids in the culture media was performed using AminoAcid Analyzer L-8900 (HITACHI). Amounts of amino acids withcorresponding values of standard deviation from data for each strain(four independent test tube fermentations) are shown in Table 4. As canbe seen in Table 4, over-expression of c0011 has positive effects onincreasing the production of valine, leucine and isoleucine in P.ananatis, which is similar to the results in E. coli for production ofall these amino acids.

TABLE 4 Strain Val (mg/L) Leu (mg/L) Ile (mg/L) SC17/pMIV-5JS  16 ± 7.22.7 ± 0.9 2.3 ± 0.3 SC17/pMIV-Pnlp0-c0011 112 ± 7.6 6.8 ± 0.3 6.0 ± 0.2

Example 4 Effect of d0663 and d0663-c09478 Locus on the FermentativeProduction of Cysteine

4-1. Construction of a Plasmid for Over-Expression of d0663 andd0663-c09478 Locus.

A DNA fragment containing approximate 200 nt downstream of d0663 ORFthrough approximate 200 nt downstream of c09478 ORF was obtained by PCRfrom genomic DNA of P. ananatis SC17 using primers P3 and P4 withPrimeSTAR DNA polymerase (Takara Bio Inc.) (5 sec at 98° C., 10 sec at55° C., 3 min at 72° C. for 30 cycles after 5 min at 94° C.). A DNAfragment containing approximate 300 nt upstream and 200 nt downstream ofthe d0663 ORF was obtained by PCR from genomic DNA of P. ananatis SC17using primers P3 and P7 (SEQ ID NO: 16) with PrimeSTAR DNA polymerase(Takara Bio Inc.) (5 sec at 98° C., 10 sec at 55° C., 3 min at 72° C.for 30 cycles after 5 min at 94° C.). The obtained 2.6 kb DNA fragmentcontaining the ORFs d0663 and c09478, and a 1.1 kb fragment containingthe d0663 ORF were cloned into pACYC177 (NIPPON GENE CO., LTD., Tokyo,Japan) using BamHI sites designed at the 5′ end of each primer to obtainplasmids pACYC-PA36 ccd and pACYC-d0663F, respectively, in which c09478on pACYC-PA36 ccd and d0663 on pACYC-d0663F, respectively, were orientedin the same direction as the Km^(r) gene located on pACYC177.

In addition to that, pMIV-Pnlp8-based expression plasmids (see Example2-1 and Example 4-2) for d0663 were constructed. A DNA fragmentcontaining P8 (SEQ ID NO: 17) and P9 (SEQ ID NO: 18) with PrimeSTAR DNApolymerase (Takara Bio Inc.) (5 sec at 98° C., 5 sec at 55° C., 90 secat 72° C. for 30 cycles after 5 min at 94° C.). Resulting DNA fragmentwas cloned into pMIV-Pnlp8 using SalI and XbaI sites designed at the 5′end of each primer to yield a plasmid pMIV-Pnlp8-d0663. Two variantclones from pMIV-Pnlp8-d0663 that confer a higher level of cysteineresistance to E. coli (see Example 1 for the resistance againstcysteine) than that with the originally constructed pMIV-Pnlp8-d0663were obtained during a course of experiments. According to thesequencing analysis of the plasmids, mutations were located not on thed0663 ORF itself but a few nucleotides upstream from the initiationcodon of d0663 raising the possibility of a translational changeproviding elevated expression level. The exact location and basesubstitution of the one mutant was C(−3)G (3^(rd) nucleotide upstreamfrom start codon “G” was substituted by “C”) and the other was C(−4)A;these variant plasmids were named pMIV-Pnlp8-d0663(−3) andpMIV-Pnlp8-d0663(−4) respectively and used as highly active expressionplasmids of d0663. These mutants can be constructed with well-knownsite-directed mutagenesis using pMIV-Pnlp8-d0663 as a start material aswell. The plasmid pMIV-5JS (JP2008-99668) was routinely used for acorresponding empty vector as a control.

4-2. Construction of the Cysteine Producing Strains.

MG1655/pACYC-E1 described in Example 2-2 was used as an E. coli straincapable of fermentative production of cysteine.

For P. ananatis cysteine-producing strains, SC17/pMIV-CysE5 andEYPSGint1M2 were prepared. Overall schematic procedures for theconstruction are described below.

The plasmid pMIV-CysE5 provides a feedback resistant mutant allele ofO-acetyl-L-serine sulfhydrylase (US20050112731A1) encoded by cysE5, anessential factor for the cysteine production. A DNA fragment containingthe cysE5 allele was obtained by PCR from pMW-PompC-cysE5 (EP1650296A1)as a template using primers P10 (SEQ ID NO: 19) and P11 (SEQ ID NO: 20)(30 sec at 94° C., 30 sec at 57° C., 1 min at 72° C. for 27 cycles andthen held at 72° C. for 7 min). The obtained 0.7 kb DNA fragmentcontaining cysE5 was cloned into pMIV-PompC using SalI and XbaI sitesdesigned at the 5′ end of each primer to yield pMIV-CysE5. The plasmidpMIV-PompC was constructed by cloning the 0.3 kb fragment containing thepromoter region of ompC gene from E. coli K-12 into pMIV-5JS(JP2008-99668) using SalI and Pad, where the fragment was obtained byPCR from genomic DNA of E. coli MG1655 using primers P12 (SEQ ID NO: 21)and P13 (SEQ ID NO: 22) and digested by SalI and Pad prior to ligation.pMIV-CysE5 was introduced into P. ananatis SC17 to provide thecysteine-producing strain SC17/pMIV-CysE5.

Construction of strain EYPSGint1M2, is described in the ReferenceExample 1.

4-3. Effect of d0663 on the Production of Cysteine in E. coli.

To test the effect of d0663 over-expression on the production ofcysteine, pMIV-Pnlp8-d0663(−3), pMIV-Pnlp8-d0663(−4) and pMIV-5JS(control) were introduced into the cysteine producing strainMG1655/pACYC-E1. Resulting strains were subjected to the productiontests comparing capacity of the fermentative production of cysteine. Theproductive cultivation was executed as described in the experiment withc0011 in E. coli (see Example 2-3) except the cultivation time was 19hrs. For maintenance of the plasmids, 25 mg/L of chloramphenicol and 20mg/L of tetracycline were supplemented during the course of cultivation.The amount of cysteine-related compounds and yield against consumedsugar with corresponding values of standard deviation from data for eachstrain (four independent test tube fermentations) are shown in Table 5.As can be seen in Table 5, over-expression of d0663 has positive effectson increasing the production of cysteine in E. coli.

TABLE 5 Strain Cysteine (mg/L) Yield (%) MG1655/pACYC-E1/pMIV-5JS 246 ±21 0.7 ± 0.07 MG1655/pACYC-E1/pMIV- 949 ± 78 2.5 ± 0.20 Pnlp8-d0663(−3)MG1655/pACYC-E1/pMIV-  980 ± 110 2.6 ± 0.23 Pnlp8-d0663(−4)

4-4. Effect of d0663 and d0663-c09478 Locus on the Production ofCysteine in P. ananatis.

To determine the effects of d0663 and d0663-c09478 locus over-expressionon the production of cysteine, pACYC-d0663F, pACYC-PA36 ccd (containstwo ORFs, d0663 and c09478) and pACYC177 (control) were introduced intothe cysteine-producing strain SC17/pMIV-CysE5. Resulting strains weresubjected to the production tests comparing capacity of the fermentativeproduction of cysteine. The productive cultivation was executed asdescribed in the experiment for cysteine production with c0011 in E.coli (see Example 2-3) except initial glucose concentration was set to60 g/L and cultivation time was 16-20 hrs. For maintenance of theplasmids, 25 mg/L of chloramphenicol and 20 mg/L of kanamycin wassupplemented into the medium during the course of cultivation. Theamount of cysteine-related compounds and yield against consumed sugarwith corresponding values of standard deviation from data for eachstrain (four independent test tube fermentations) are shown in Table 6.As can be seen in Table 6, over-expression of d0663 has positive effectson increasing the production of cysteine in P. ananatis. It was foundthat addition of c09478 accelerated the effects of d0663 as pACYC-PA36ccd doubled the production of cysteine as compared to the productionwith pACYC-d0663F.

TABLE 6 Strain Cysteine (mg/L) Yield (%) SC17/pMIV-CysE5/pACYC177 200 ±62 0.3 ± 0.10 SC17/pMIV-CysE5/pACYC-d0663F 496 ± 75 0.8 ± 0.13SC17/pMIV-CysE5/pACYC-PA36ccd 906 ± 96 1.5 ± 0.16

Same experiments were performed using EYPSGint1M2 as a cysteine-producerstrain. Again, the plasmids pACYC-d0663F, pACYC-PA36 ccd (contains twoORFs, d0663 and c09478) and pACYC177 (control) were introduced, and theresulting strains were subjected to the production tests described inthe experiment for cysteine production with c0011 in E. coli (seeExample 2-3). For maintenance of the plasmids, 20 mg/L of kanamycin wassupplemented during the course of cultivation. The amount ofcysteine-related compounds and yield against consumed sugar withcorresponding values of standard deviation from data for each strain(four independent test tube fermentations) was shown in Table 7. As canbe seen in Table 7, over-expression of d0663 has positive effects onincreasing the production of cysteine in P. ananatis, which was furtherpromoted by the addition of c09478.

TABLE 7 Strain Cysteine (g/L) Yield (%) EYPSGint1M2/pACYC177 0.55 ± 0.101.4 ± 0.24 EYPSGint1M2/pACYC-d0663F 0.96 ± 0.15 2.4 ± 0.37EYPSGint1M2/pACYC- 1.34 ± 0.20 3.4 ± 0.49 PA36ccd

Example 5 Effects of d0663 on the Fermentative Production of Amino Acids

5-1. Effect of d0663 on the Production of Amino Acids in P. ananatis.

To determine the effects of d0663 over-expression on the production ofamino acids, pMIV-Pnlp8-d0663(−3), pMIV-Pnlp8-d0663(−4) and pMIV-5JS(control) were introduced into P. ananatis SC17. Resulting strains weresubjected to the production tests comparing capacity of the fermentativeproduction of amino acids. The productive cultivation was executed asdescribed in the experiment for cysteine production with c0011 in E.coli (see Example 2-3) except the initial glucose concentration was setto 60 g/L and cultivation time was 18 hrs. For maintenance of theplasmids, 25 mg/L of chloramphenicol was supplemented during the courseof cultivation. Quantitative analysis of produced amino acids in theculture media was performed using Amino Acid Analyzer L-8900 (HITACHI).Amount of amino acids with corresponding values of standard deviationfrom data for each strain (four independent test tube fermentation) areshown in Table 8. As can be seen in Table 8, over-expression of d0663has positive effects on increasing the production of valine, leucine,isoleucine and threonine.

TABLE 8 Ile Thr Strain Val (mg/L) Leu (mg/L) (mg/L) (mg/L) SC17/pMIV-5JS105.6 ± 9.3  5.8 ± .5 3.7 ± 0.3  2.1 ± 0.1 SC17/pMIV- 1286.2 ± 21.0111.7 ± 6.9 61.8 ± 3.5 58.4 ± 0.1 Pnlp8-d0663 (-3) SC17/pMIV- 1367.1 ±27.1 134.0 ± 5.8 67.1 ± 2.7 62.2 ± 3.4 Pnlp8-d0663 (-4)

Example 6 Effect of Putative Cys Exporters on the FermentativeProduction of L-Valine by E. coli

6-1. Effect of d0663(−4) on the Production of L-Valine in E. coli

To test effect of d0663(−4) over-expression on the production of valine,pMIV-Pnlp8-d0663 (−4) and pMIV-5JS (control) plasmids were introducedinto the L-valine producing strain H-81. The strain H-81 has beendeposited in the Russian National Collection of IndustrialMicroorganisms (VKPM) (Russia 113545, Moscow, 1 Dorozhny proezd, 1) onJan. 30, 2001 under accession number VKPM B-8066, and converted to aninternational deposit based on Budapest Treaty on Feb. 1, 2002.Resulting strains were subjected to the production tests comparingcapacity of the fermentative production of L-valine. Cells from stocktube (stored in 25% glycerol, 0.9% NaCl at −70° C.) were plated onL-agar (yeast extract—5 g/l, peptone—10 g/l, NaCl—5 g/l, agar—15 g/l).For plasmid strains L-agar was supplemented with appropriate antibiotic(Ap—100 mg/l, Km—40 mg/l). Cells from about 0.5 cm² of plate surfacewere inoculated into fermentation medium (2 ml) and cultivated for 72hours at 32° C. The composition of the fermentation medium was asfollows: sucrose—60 g/l, (NH₄)₂SO₄—15 g/l, KH₂PO₄—1.5 g/l, MgSO₄—1 g/l,thiamin—0.1 g/l, Mameno (TN)—0.4 g/l, CaCO₃—25 g/l, pH 7.0 (KOH),appropriate antibiotics (Km, 50 mg/l; Ap, 100 mg/l) were added into themedium.

Quantitative analysis of produced valine in the culture medium wasperformed using Amino Acid Analyzer L-8900 (HITACHI). Amount of valinewith corresponding values of standard deviation from data for eachstrain (four independent test tube fermentations) are shown in Table 9.As can be seen in Table 9, over-expression of d0663(−4) have positiveeffects on increasing the production of L-valine by E. coli.

TABLE 9 Strain Val (g/L) OD₅₄₀ H-81/pMIV-5JS 11.2 ± 0.3 33.2 ± 1.2H-81/pMIV-Pnlp8-d0663 (-4) 13.2 ± 1.2 28.7 ± 1.2

6-2. Effect of c0011 on the Production of L-Valine in E. coli

To test effect of c0011 overexpression on the production of L-valine,the pMIV-Pnlp23-c0011 plasmid was introduced into the valine-producingstrain H-81. Resulting strain together with parent H-81 strain wassubjected to the production tests comparing capacity of the fermentativeproduction of L-valine using fed-batch cultivation in Marubishifermentors.

Cells were plated on L-agar (yeast extract—5 g/l, peptone—10 g/l, NaCl—5g/l, agar—15 g/l). For plasmid strain, L-agar was supplemented withappropriate antibiotic (Ap—100 mg/l, Km—40 mg/l). Cells from about 0.5cm² of plate surface were inoculated into L-broth (tryptone—10 g/l,yeast extract—5 g/l, NaCl—5 g/l) medium (60 ml) and cultivated for 20hours at 37° C. at 240 rpm. Then 40 ml of seed culture were transferredto 360 ml of fermentation medium in jar-fermentor and cultivated for 20hours at 37° C. initially with agitation at 1200 rpm and after 18 hoursof cultivation at 900 rpm.

The composition of the fermentation medium was as follows: glucose—30g/l, MgSO₄ 7H₂O—0.4 g/l, Mameno (TN)—0.4 g/l, (NH₄)₂SO₄—5.0 g/l,KH₂PO₄—3.0 g/l, FeSO₄ 7H₂O—0.02 g/l, MnSO₄ 5H₂O—0.02 g/l, thiamin—0.4mg/l, antifoam—0.1 mg/l, pH 6.6 (KOH). Mixed feed (glucose—140 g,H₂O—100 ml, concentrate aqueous ammonia—53 ml) was added automaticallyto maintain a constant pH of 6.6.

The data on production of valine are presented in Table 10. As can beseen in Table 10, over-expression of c0011 has positive effect onincreasing the production of valine in E. coli.

TABLE 10 Val Strain Y, % OD g/l g/l h H-81 24.3 133 41.1 2.0H-81/pMIV-Pnlp23-c0011 26.3 98 50.6 2.5

Example 7 Effects of d0663 on the Production of O-acetyl-L-serine in E.coli

To determine effects of d0663 over-expression on the production ofO-acetyl-L-serine, pMIV-Pnlp8-d0663(−3) and pMIV-5JS (control) plasmidswere introduced into the cysteine-producing strain MG1655/pACYC-E1.Since the strain MG1655/pACYC-E1 carries feed-back resistant CysE mutanton the plasmid, this strain is suitable for producing O-acetyl-L-serine.Resulting strains were subjected to the production tests comparingcapacity of the fermentative production of O-acetyl-L-serine. Theproductive cultivation was executed as described in the experiment withc0011 in E. coli (see Example 2) except cultivation time was 26 hours.For maintenance of the plasmids, 25 mg/L of chloramphenicol and 12.5mg/L of tetracycline were supplemented during the course of cultivation.In order to convert produced O-acetyl-L-serine to N-acetylserine (NAS)at alkaline pH, samples were diluted using 200 mM Tris/HCl (pH9.0).Resulting NAS was analyzed using HPLC. The analytic conditions for HPLCwere the following: column: Inertsil ODS-3 (GL science), flow rate: 1.0ml/min, column temperature: 40° C., detector: UV210 mm, sample volume:10:1, buffer: 0.1M K₂PO₄—H₃PO₄ (pH2.2) 5 mM sodium 1-octanesulfonate.Amount of O-acetyl-L-serine and yield against consumed sugar withcorresponding values of standard deviation from triplicate data for eachstrain was shown in Table 11. The obtained data suggests thatover-expression of d0663 has positive effects on increasing theproduction of O-acetyl-L-serine in E. coli.

TABLE 11 O-acetyl-L-serine Strain (g/L) Yield (%)MG1655/pACYC-E1/pMIV-5JS 0.20 ± 0.20 0.60 ± 0.51MG1655/pACYC-E1/pMIV-Pnlp8-  4.1 ± 0.34 10.3 ± 0.86 d0663 (-3)

Example 8 Effects of c0011 on the Production of O-acetyl-L-serine in E.coli

To determine effects of c0011 over-expression on the production ofO-acetyl-L-serine, pMIV-Pnlp23-c0011 and pMIV-5JS (control) plasmidswere introduced into the cysteine/O-acetyl-L-serine producing strainMG1655/pACYC-E1. All the productive cultivation and analytic procedureswere the same as described in the production of O-acetyl-L-serine withd0663 enhanced strain (see example 7). Amount of producedO-acetyl-L-serine and yield against consumed sugar with correspondingvalues of standard deviation from triplicate data for each strain wasshown in Table 12. The obtained data suggests that over-expression ofc0011 has positive effects on increasing the production ofO-acetyl-L-serine in E. coli.

TABLE 12 O-acetyl-L- Strain serine (g/L) Yield (%)MG1655/pACYC-E1/pMIV-5JS 0.60 ± 0.04 1.49 ± 0.11MG1655/pACYC-E1/pMIV-Pnlp23-c0011 2.46 ± 0.86 6.16 ± 2.15

Reference Example 1 Construction of a Strain with Enhanced Expression ofthe Gene cysM

1. Construction of the Strain P. ananatis EYPS1976(s)

The DNA fragment containing promoter of the gene nlpD from E. coli wasobtained using PCR. The chromosomal DNA of E. coli MG1655 (ATCC 700926)strain as a template and primers P14 (SEQ ID No:23) and P15 (SEQ IDNo:24) were used for PCR. Primer P14 (SEQ ID No:23) contains a site forSalI restrictase at the 5′-end thereof. Primer P15 (SEQ ID No:24)contains a site for PaeI restrictase at the 5′-end thereof. Conditionsfor PCR were as follows: denaturation step for 3 min at 95° C.; profilefor two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72°C.; profile for the last 25 cycles: 20 sec at 94° C., 20 sec at 55° C.,15 sec at 72° C.; final step: 5 min at 72° C. The amplified DNA fragmentwas about 0.2 kb in size, it was purified by agarose gelelectrophoresis. Then, the purified fragment was treated withendonucleases Pad and SalI. The obtained DNA fragment was then ligatedwith plasmid pMIV-5JS (construction of the plasmid pMIV-5JS is describedin EP1942183B1) previously treated with endonucleases Pad and SalI. Themixture for ligation was incubated at 4° C. overnight and was then usedto transform E. coli strain MG1655 by electroporation. The transformantswere plated on plates with LB agar containing ampicillin (50 mg/l), theplates were incubated at 37° C. overnight until individual coloniesbecame visible. Plasmids were isolated from obtained transformants andanalyzed by restriction analysis. The obtained plasmid containing thepromoter of the gene nlpD from E. coli was named pMIV-Pnlp0.

The DNA fragment containing terminator of the gene rrnB from E. coli wasobtained using PCR. The chromosomal DNA of E. coli MG1655 strain as atemplate and primers P16 (SEQ ID No:25) and P17 (SEQ ID No:26) were usedfor PCR. Primer P16 (SEQ ID No:25) contains a site for XbaI restrictaseat the 5′-end thereof. Primer P17 (SEQ ID No:26) contains a site forBamHI restrictase at the 5′-end thereof. Conditions for PCR were asfollows: denaturation step for 3 min at 95° C.; profile for two firstcycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile forthe last 25 cycles: 20 sec at 94° C., 20 sec at 59° C., 15 sec at 72°C.; final step: 5 min at 72° C. The amplified DNA fragment was about 0.3kb in size, and it was purified by agarose gel electrophoresis. Then,the purified fragment was treated with endonucleases BamHI and XbaI. Theobtained DNA fragment was then ligated with plasmid pMIV-Pnlp0previously treated with endonucleases BamHI and XbaI. The mixture forligation was incubated at 4° C. overnight and was then used to transformE. coli strain MG1655 by electroporation. The transformants were platedon plates with LB agar containing ampicillin (50 mg/l), the plates wereincubated at 37° C. overnight until individual colonies became visible.Plasmids were isolated from obtained transformants and analyzed byrestriction analysis. The obtained plasmid containing the terminator ofthe gene rrnB from E. coli was named pMIV-Pnlp0-ter.

The DNA fragment containing the gene yeaS from E. coli was obtainedusing PCR. The chromosomal DNA of E. coli MG1655 strain as a templateand primers P18 (SEQ ID No:27) and P19 (SEQ ID No:28) were used for PCR.Primer P18 (SEQ ID No:27) contains a site for SalI restrictase at the5′-end thereof. Primer P19 (SEQ ID No:28) contains a site for XbaIrestrictase at the 5′-end thereof. Conditions for PCR were as follows:denaturation step for 3 min at 95° C.; profile for two first cycles: 1min at 95° C., 30 sec at 50° C., 40 sec at 72° C.; profile for the last25 cycles: 20 sec at 94° C., 20 sec at 55° C., 15 sec at 72° C.; finalstep: 5 min at 72° C. The amplified DNA fragment was about 0.7 kb insize, and it was purified by agarose gel electrophoresis. Then, thepurified fragment was treated with endonucleases SalI and XbaI. Theobtained DNA fragment was then ligated with plasmid pMIV-Pnlp0-terpreviously treated with endonucleases SalI and XbaI. The mixture forligation was incubated at 4° C. overnight and was then used to transformE. coli strain MG1655 by electroporation. The transformants were platedon plates with LB agar containing ampicillin (50 mg/l), the plates wereincubated at 37° C. overnight until individual colonies became visible.Plasmids were isolated from obtained transformants and analyzed byrestriction analysis. The obtained plasmid containing the gene yeaS fromE. coli was named pMIV-Pnlp0-yeaS3.

Then randomization of −10 region of promoter Pnlp and selection of thePnlp8 promoter was performed. The 3′-end of promoter Pnlp was obtainedusing PCR amplification. The plasmid pMIV-Pnlp0 as a template andprimers P14 (SEQ ID No:23) and P20 (SEQ ID No:29) were used for PCR.Primer P20 has random nucleotides, depicted in SEQ ID NO: 29 by theletter “n” (for A or G or C or T) and site for BglII restrictase at the5′-end thereof. Conditions for PCR were as follows: denaturation stepfor 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30sec at 50° C., 40 sec at 72° C.; profile for the last 25 cycles: 20 secat 94° C., 20 sec at 60° C., 15 sec at 72° C.; final step: 5 min at 72°C. 5′-end of promoter Pnlp was obtained using PCR amplification. Theplasmid pMIV-Pnlp0 as a template and primers P15 (SEQ ID No:24) and P21(SEQ ID No:30) were used for PCR. Primer P21 has random nucleotides,depicted in SEQ ID NO: 30 by the letter “n” (for A or G or C or T) andsite for BglII restrictase at the 5′-end thereof. Conditions for PCRwere as follows: denaturation step for 3 min at 95° C.; profile for twofirst cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72° C.;profile for the last 25 cycles: 20 sec at 94° C., 20 sec at 60° C., 15sec at 72° C.; final step: 5 min at 72° C. Both amplified DNA fragmentswere purified by agarose gel electrophoresis. Then, the obtained DNAfragments were treated with endonuclease BglII followed by ligation ofthe fragments in equimolar proportion. The mixture for ligation wasincubated at 4° C. overnight and was then used as a template for nextPCR procedure, primers P14 (SEQ ID No:23) and P15 (SEQ ID No:24) wereused for the PCR. Conditions for PCR were as follows: denaturation stepfor 3 min at 95° C.; profile for two first cycles: 1 min at 95° C., 30sec at 50° C., 40 sec at 72° C.; profile for the last 12 cycles: 20 secat 94° C., 20 sec at 60° C., 15 sec at 72° C.; final step: 5 min at 72°C.

The amplified DNA fragment was about 0.2 kb in size, and it was purifiedby agarose gel electrophoresis. Then, the purified fragment was treatedwith endonucleases PaeI and SalI. The obtained DNA fragment was thenligated with plasmid pMIV-Pnlp0-yeaS3 previously treated withendonucleases PaeI and SalI. The mixture for ligation was incubated at4° C. overnight and was then used to transform E. coli strain MG1655 byelectroporation. The transformants were plated on plates with LB agarcontaining ampicillin (50 mg/l), the plates were incubated at 37° C.overnight until individual colonies became visible. Plasmids wereisolated from the obtained transformants and analyzed by sequencinganalysis. The obtained plasmid containing promoter Pnlp8 was namedpMIV-Pnlp8-yeaS7.

Then, the plasmid pMIV-Pnlp8-yeaS7 was treated with endonuclease HindIIIfollowed by purification and treatment with DNA polymerase I largefragment (Klenow fragment). The obtained DNA fragment was purified andtreated with endonuclease NcoI.

The obtained DNA fragment after purification was then ligated inequimolar proportion with plasmid pMT-Pomp-cysE5 (pMT-Pomp-cysE5 wasderived from pMIV-Pomp-cysE5 by cloning of XbaI-Eco88I/Klenow fragmentfrom pACYC-184 (tet-R) into PvuI site of pMIV-Pomp-cysE5.pMIV-Pomp-cysE5 was obtained by subcloning PaeI+SacI fragment frompMW-Pomp-cysE5 (WO2005007841) into the same sites of pMIV-5JS)previously treated with endonucleases SmaI and NcoI. The mixture forligation was incubated at 4° C. overnight and was then used to transformE. coli strain MG1655 by electroporation. The transformants were platedon plates with LB agar containing ampicillin (50 mg/l), the plates wereincubated at 37° C. overnight until individual colonies became visible.Plasmids were isolated from obtained transformants and analyzed byrestriction analysis. The obtained plasmid containing cysE5 was namedpMT-EY2. Enzymatic activity of serine acetyltransferase was measured inobtained transformant in order to confirm intactness of cysE5 allele.

The next step was to integrate cysE5 and yeaS genes into the chromosomeof P. ananatis strain SC17 (U.S. Pat. No. 6,596,517). Plasmid pMH10(Zimenkov D. et al., Biotechnology (in Russian), 6, 1-22 (2004)) wasused to transform P. ananatis strain SC17 by electroporation. Thetransformants were plated on plates with LB agar containing kanamycin(20 mg/l), the plates were incubated at 30° C. overnight untilindividual colonies became visible. The obtained strain SC17/pMH10 wasreseeded two times. After that, plasmid pMT-EY2 was used to transform P.ananatis strain SC17/pMH10 (this strain was grown at 30° C.) byelectroporation. The transformants were shocked by incubation at hightemperature (42° C., 20 min) and plated on plates with LB agarcontaining chloramphenicol (20 mg/l); the plates were incubated at 39°C. overnight until individual colonies became visible. About 50 cloneswere reseeded at 39° C. and each of them were inoculated in 1 ml of LAmedium and incubated at 39° C. for 48 h. After incubation all 50variants were tested for curing of the plasmids pMH10 and pMT-EY2,variants were selected which were resistant to chloramphenicol (20 mg/l)but sensitive to kanamycin (20 mg/l) and ampicillin (50 mg/l). Desiredintegrants were identified by PCR analysis using primers P1 and P6. Theobtained line of strains was named as EY01-EY50 and all of them weretested for their ability to produce cysteine in test-tube fermentation.The best producer strain EY19 was selected and used in the followingexperiments.

To cure the P. ananatis strain EY19 from resistance to chloramphenicol,strain EY19 was transformed with the plasmid pMT-Int-Xis2(WO2005/010175) using electroporation. The transformants were plated onplates with LB agar containing tetracycline (10 mg/l); the plates wereincubated at 30° C. overnight until individual colonies became visible.The objective transformants were identified by selecting variants whichwere sensitive to chloramphenicol (20 mg/l). The “cured” strain wasnamed EY19(s).

The next step was to substitute the promoter region of cysPTWA geneswith the Pnlp8 promoter region in the strain EY19(s). PCR was carriedout using the plasmid pMIV-Pnlp8-yeaS7 as a template and primers P14 andP15. Conditions for PCR were as follows: denaturation step for 3 min at95° C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C.,40 sec at 72° C.; profile for the last 20 cycles: 20 sec at 94° C., 20sec at 59° C., 15 sec at 72° C.; final step: 5 min at 72° C. Theamplified DNA fragment was about 0.2 kb in size, and it was purified byagarose gel electrophoresis. Then, the purified fragment was treatedwith Klenow fragment. The DNA fragment was then ligated in equimolarproportion with plasmid pMW118-(XattL-Km^(r)-XattR) (EP2100957A1)previously treated with endonuclease XbaI followed by treatment withKlenow fragment. The mixture for ligation was incubated at 4° C.overnight and was then used to transform E. coli strain MG1655 byelectroporation. The transformants were plated on plates with LB agarcontaining kanamycin (20 mg/l), the plates were incubated at 37° C.overnight until individual colonies became visible. Plasmids wereisolated from the transformants and analyzed by restriction analysis.The obtained plasmid containing the Pnlp promoter was namedpMW-Km-Pnlp8. Then, PCR was carried out using the plasmid pMW-Km-Pnlp8as a template and primers P22 (SEQ ID No:31) and P23 (SEQ ID No:32).Conditions for PCR were as follows: denaturation step for 3 min at 95°C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40sec at 72° C.; profile for the last 30 cycles: 20 sec at 94° C., 20 secat 54° C., 90 sec at 72° C.; final step: 5 min at 72° C. The obtainedDNA fragment was about 1.6 kb in size, and it was purified by agarosegel electrophoresis and used to transform P. ananatis strain SC17(0) byelectroporation. The transformants were plated on plates with LB agarcontaining kanamycin (20 mg/l); the plates were incubated at 34° C.overnight until individual colonies became visible. The objectivetransformants were identified by PCR analysis using primers P24 (SEQ IDNo:33) and P25 (SEQ ID No:34). The obtained strain was namedSC17-Pnlp8-PTWA. Chromosomal DNA was isolated from the strainSC17-Pnlp8-PTWA. 10 μg of this chromosomal DNA was used to transform P.ananatis strain EY19(s) by electroporation. The transformants wereplated on plates with LB agar containing kanamycin (20 mg/l), the plateswere incubated at 34° C. overnight until individual colonies becamevisible. The transformants were identified by PCR analysis using primersP24 and P25. The obtained strain was named EYP197. To cure the P.ananatis strain EYP197 from resistance to kanamycin strain, EYP197 wastransformed with the plasmid pMT-Int-Xis2 by electroporation. Thetransformants were plated on plates with LB agar containing tetracycline(10 mg/l); the plates were incubated at 30° C. overnight untilindividual colonies became visible. The objective transformants wereidentified by selecting variants which were sensitive to kanamycin (20mg/l). The “cured” strain was named EY197(s).

Mutation N348A was introduced by site-specific mutagenesis. For thispurpose, the 3′-end of gene serA (with mutation) was obtained by PCRamplification using chromosomal DNA of the strain SC17 as a template andprimers P26 (SEQ ID No:35) and P27 (SEQ ID No:36), and the 5′-end ofserA gene was obtained by PCR amplification using the chromosomal DNA ofthe strain SC17 as a template and primers P28 (SEQ ID No:37) and P29(SEQ ID No:38). Both primer P27 (SEQ ID No:36) and P29 (SEQ ID No:38)contain a site for SmaI restrictase at the 5′-end thereof. Conditionsfor the first PCR were as follows: denaturation step for 3 min at 95°C.; profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40sec at 72° C.; profile for the last 25 cycles: 20 sec at 94° C., 20 secat 60° C., 60 sec at 72° C.; final step: 5 min at 72° C. Conditions forthe second PCR were as follows: denaturation step for 3 min at 95° C.;profile for two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 secat 72° C.; profile for the last 20 cycles: 20 sec at 94° C., 20 sec at60° C., 20 sec at 72° C.; final step: 5 min at 72° C. Both amplified DNAfragments were purified by agarose gel electrophoresis followed bytreatment with endonuclease SmaI. The obtained DNA fragments were thenligated in equimolar proportion. The mixture for ligation was incubatedat 4° C. overnight and was used as a template for the next PCR procedure(with primers P26 and P28). Primer P26 (SEQ ID No:35) contains a sitefor SalI restrictase at the 5′-end thereof. Primer P28 (SEQ ID No:37)contains a site for XbaI restrictase at the 5′-end thereof. Conditionsfor PCR were as follows: denaturation step for 3 min at 95° C.; profilefor two first cycles: 1 min at 95° C., 30 sec at 50° C., 40 sec at 72°C.; profile for the last 15 cycles: 20 sec at 94° C., 20 sec at 60° C.,75 sec at 72° C.; final step: 5 min at 72° C. The amplified DNA fragmentwas about 1.3 kb in size, and it was purified by agarose gelelectrophoresis. The obtained fragment was treated with endonucleasesSalI and XbaI. After restriction DNA fragment was ligated in equimolarproportion with the plasmid pMIV-Pnlp8-ter previously treated withendonucleases SalI and XbaI. The mixture for ligation was incubated at4° C. overnight and was then used to transform E. coli strain MG1655 byelectroporation. The transformants were plated on plates with LB agarcontaining ampicillin (50 mg/l), the plates were incubated at 37° C.overnight until individual colonies became visible. Plasmids wereisolated from the transformants and analyzed by sequencing analysis. Theobtained plasmid containing serA gene with mutation N348A was namedpMIV-Pnlp8-serA348.

The next step was to integrate serA348 allele into chromosome of the P.ananatis strain SC17. Plasmid DNA pMIV-Pnlp8-serA348 was used totransform P. ananatis strain SC17/pMH10 (this strain was grown at 30°C.) by electroporation. The transformants were shocked by incubation athigh temperature (42° C., 20 min) and plated on plates with LB agarcontaining chloramphenicol (20 mg/l), the plates were incubated at 39°C. overnight until individual colonies became visible. About 50 cloneswere reseeded at 39° C. and then each of them were inoculated in 1 ml ofLA medium and incubated at 39° C. for 48 h. After incubation all 50variants were tested for curing of the plasmids pMH10 andpMIV-Pnlp8-serA348 by selecting variants which were resistant tochloramphenicol (20 mg/l) but sensitive to kanamycin (20 mg/l) andampicillin (50 mg/l). The objective integrants were identified by PCRanalysis using primers P14 and P28. In all the obtained variants, thespecific activity of PGD was measured and the most active one wasselected for the following purpose. It was named SC17int-serA348.

The next step was to transfer an integrated copy of serA348 into thestrain EYP197(s).

Chromosome DNA was isolated from the strain SC17int-serA348. 10 μg ofthis chromosomal DNA was used to transform P. ananatis EYP197(s) byelectroporation. The transformants were plated on plates with LB agarcontaining chloramphenicol (20 mg/l), the plates were incubated at 34°C. overnight until individual colonies became visible. The objectivetransformants were identified by PCR analysis using primers P14 and P28.This strain was named EYPS1976.

To cure the P. ananatis strain EYPS1976 from resistance tochloramphenicol, strain EYPS1976 was transformed with the plasmidpMT-Int-Xis2 by electroporation. The transformants were plated on plateswith LB agar containing tetracycline (10 mg/l), and the plates wereincubated at 30° C. overnight until individual colonies became visible.The objective transformants were identified by selecting variants whichwere sensitive to chloramphenicol (20 mg/l). This “cured” strain wasnamed EYPS1976(s).

2. Construction of the Strain EYPSGint1M2

The strain EYPSGint1M2 was obtained by integration of the cysM geneencoding O-acetylserine (thiol)-lyase B from E. coli into chromosome ofEYPS1976(s). At the first step, integration of cysM was obtained as aresult of mini-μ integration of the plasmid pMIV-Pnlp1-cysM (Referenceexample 2) into the chromosome of the strain SC17 using μ-transposasecarrying plasmid pMH10 (as described above). The obtained construction(int(Pnlp1-cysM)) was transferred from the strain SC17 into the strainEYPS1976(s) by a chromosome transformation procedure using Cm^(R) markerfor selection. This strain was named as EYPSGint1M2 and after curingfrom antibiotic resistance marker, it was used for further experiments.

Reference Example 2 Construction of the Plasmid pMIV-Pnlp1-cysM

The promoter region of the gene nlpD from P. ananatis was amplified byPCR using primers P30 (SEQ ID NO: 39) and P31 (SEQ ID NO: 40) andchromosomal DNA of the strain SC17 as a template. The obtained DNAfragment was about 0.2 kb in size, and it was purified by agarose gelelectrophoresis and digested with PaeI and SalI endonucleases followedby ligation with plasmid pMIV-5.TS, previously treated with the sameendonucleases.

The region of the terminator of the E. coli gene rrnB was amplified byPCR using primers P32 (SEQ ID NO: 41) and P33 (SEQ ID NO: 42) andchromosomal DNA of the strain MG1655 as a template. The obtained DNAfragment was about 0.25 kb in size, and it was purified by agarose gelelectrophoresis and digested with XbaI and BamHI endonucleases followedby ligation with obtained at the previous step plasmid, previouslytreated with the same endonucleases.

The E. coli gene cysM was amplified by PCR using primers P34 (SEQ ID NO:43) and P35 (SEQ ID NO: 44) and chromosomal DNA of the strain MG1655 asa template. The obtained DNA fragment was about 1.1 kb in size, and itwas purified by agarose gel electrophoresis and digested with SalI andXbaI endonucleases followed by ligation with the plasmid obtained at theprevious step, previously treated with the same endonucleases. Thus, theplasmid pMIV-Pnlp1-cysM was obtained.

While the invention has been described in detail with reference topreferred embodiments thereof, it will be apparent to one skilled in theart that various changes can be made, and equivalents employed, withoutdeparting from the scope of the invention. Each of the aforementioneddocuments is incorporated by reference herein in its entirety.

1. A method for producing an L-amino acid comprising: A) cultivating anEnterobacteriaceae bacterium that is able to produce an L-amino acid ina culture medium, and B) collecting the L-amino acid from the culturemedium or the bacterium, wherein the bacterium has been modified toincrease an activity of a protein which is able to confer to thebacterium resistance to growth inhibition by L-cysteine as compared to anon-modified bacterium, wherein said protein is selected from the groupconsisting of: (A) the protein of SEQ ID NO: 2 or a variant thereof, and(B) the protein of SEQ ID NO: 4 or a variant thereof.
 2. The methodaccording to claim 1, wherein expression of a DNA encoding said proteinin said bacterium is enhanced.
 3. The method according to claim 1,wherein said bacterium is transformed with a DNA encoding said protein.4. The method according to claim 2, wherein the DNA comprises a geneselected from the group consisting of c0011 and d0663.
 5. The methodacceding to claim 1, wherein said protein is at least the protein (B) ora variant thereof, and said bacterium has been further modified toincrease expression of a DNA encoding the protein of SEQ ID NO: 6 or avariant thereof.
 6. The method according to claim 5, wherein the DNA isthe c09478 gene.
 7. The method according to claim 1, wherein thebacterium belongs to the genus Escherichia.
 8. The method according toany of claims 1, wherein the bacterium belongs to the genus Pantoea. 9.The method according to claim 7, wherein the bacterium is Escherichiacoli.
 10. The method according to claim 8, wherein the bacterium isPantoea ananatis.
 11. The method according to claims 1, wherein saidL-amino acid is selected from the group consisting of an aromaticL-amino acid and a non-aromatic L-amino acid.
 12. The method accordingto claim 11, wherein said aromatic L-amino acid is selected from thegroup consisting of L-phenylalanine, L-tyrosine, and L-tryptophan. 13.The method according to claim 12, wherein said non-aromatic L-amino acidis selected from the group consisting of L-threonine, L-lysine,L-cysteine and L-cysteine derivatives, L-methionine, L-leucine,L-isoleucine, L-valine, L-histidine, glycine, L-serine, L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, L-proline,L-arginine and O-acetyl-L-serine.
 14. The method according to claim 11,wherein said L-amino acid is selected from the group consisting ofL-cysteine, L-valine, L-leucine, L-Isoleucine, L-threonine, L-glutamicacid, L-glycine, L-alanine, L-histidine, and O-acetyl-L-serine.